Genomic approach to identification of novel broad-spectrum antimicrobial peptides from bony fish

ABSTRACT

There is provided a method of identifying candidate nucleic acid sequences encoding antimicrobial peptides. The method comprises: identifying an initial peptide of interest; identifying genomic DNA encoding the initial peptide; identifying a flanking sequence on each side of the initial peptide; obtaining primers complementary to the flanking sequences; and, screening a wide range of nucleic acid sequences to identify candidate sequences capable of being amplified using the primers from step e). In some instances the antimicrobial peptide is a hepcidin or a pleurocidin.

The present application is a 371 of PCT/CA2003/001323 and claimspriority under 35 U.S.C. §119 to U.S. Application Ser. No. 60/404,922filed Aug. 22, 2002.

BACKGROUND OF THE INVENTION

Antimicrobial peptides have been isolated from a wide variety of plantsand animals, and play an important role in defense against microbialinvasion. They fall into three main classes based on secondary structureand amino acid sequence similarities: α-helical structures, highlydisulphide-bonded (cysteine-rich) β-sheets and those with a highpercentage of single amino acids such as proline or arginine. Mostmolecules are amphiphilic and contain both cationic and hydrophobicsurfaces, enabling them to insert into biological membranes. Althoughone of the modes of action of antimicrobial peptides has been describedas lysis of pathogens, they may also exert their effects by binding tointracellular targets. They have also been reported to exert a number ofeffects such as mediating inflammation and modulating the immuneresponse.

A small number of natural antimicrobial peptides have been isolated fromteleosts including the pleurocidin, from the skin of winter flounder(Cole, Weis et al. 1997), pardaxin from Red Sea Moses sole (Oren andShai 1996), misgumin from loach (Park, Lee et al. 1997), HFA-1 fromhagfish (Hwang, Seo et al. 1999), piscidins from hybrid striped basseosinophilic granule cells (Silphaduang and Noga 2001), moronecidinsfrom hybrid striped bass (Lauth, Shike et al. 2002), parasin, a cleavageproduct of histone 2A from catfish (Park, Park et al. 1998) and someuncharacterized mucous secretions from carp (LeMaitre, Orange et al.1996) and trout (Smith, Fernandes et al. 2000). In addition, a cationicsteroidal antibiotic, squalamine, has been isolated from the shark,Squalus acanthias (Moore, Wehrli et al. 1993).

Cysteine-rich antimicrobial peptides of the defensin family have beendetected in the fat body of insects and the hemolymph of molluscs andcrustaceans. They have also been isolated from various epithelia ofmammals as well as circulating cells such as neutrophils andmacrophages. Recently, small cysteine-rich peptides exhibitingantimicrobial activity against various fungi, Gram positive and Gramnegative bacteria have been isolated from blood ultrafiltrate (Krause,Neitz et al. 2000), the human urinary tract (Park, Valore et al. 2001),and the gill of bacterially challenged hybrid striped bass (Shike et al.2002). These peptides, referred to as hepcidin or LEAP-1(liver-expressed antimicrobial peptide), have been proposed to be thevertebrate counterpart of insect peptides induced in the fat body inresponse to infection (Park, Valore et al. 2001). Antimicrobial peptideshave a variety of potential uses. (see for example U.S. Pat. No.6,288,212 of Hancock)

The conventional approach to identifying antimicrobial peptides involvesbiochemical purification from tissues or secretions. Fractions aretested for antimicrobial activity, and the purified peptides thatexhibit activity are then sequenced. This approach is costly, timeconsuming, and not well suited to the identification of low abundance ordifficult-to-purify antimicrobial peptides.

Thus, it is an object of the invention to provide a method foridentifying potential antimicrobial peptides.

SUMMARY OF THE INVENTION

In one aspect of the invention there is provided a method of identifyingcandidate nucleic acid sequences encoding antimicrobial peptides, saidmethod comprising:

-   -   (a) identifying an initial peptide of interest;    -   (b) identifying genomic DNA encoding the initial peptide;    -   (c) identifying a flanking sequence on each side of the initial        peptide;    -   (d) obtaining primers complementary to the flanking sequences;        and,    -   (e) screening a wide range of nucleic acid sequences to identify        candidate sequences capable of being amplified using the primers        from step d).

According to one aspect of the invention the nucleotide and deducedamino acid sequences of hepcidin-like peptides are provided.

According to another aspect of the invention, the nucleotide and deducedamino acid sequences of pleurocidin-like peptides are provided.

According to another aspect of the invention primers suitable for use inthe identification, isolation and/or amplification of nucleic acidsequences encoding novel microbial peptides are provided.

According to another aspect of the invention there is provided a methodfor the identification of families of nucleic acid sequences encodingantimicrobial peptides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a textual and graphical depiction of pleurocidin WF2 cDNA fromwinter flounder (A), a graphical depiction of a predicted hydrophobicityplot of peptide WF2 (B), and a diagrammatic depiction of a predictedhelical structure of WF2 (C).

FIG. 2 is a pictorial depiction of results of amplification of certainhepcidin-like cDNAs.

FIG. 3 is a depiction of certain aligned pleurocidin-like peptidesequences.

FIG. 4 is a pictorial depiction of the results of PCR amplification ofcertain pleurocidin-like genomic sequences.

FIG. 5 is a depiction of an extended genomic sequence of WF4.

FIG. 6 is a depiction of an alignment of certain pleurocidin-likepolypeptide sequences.

FIG. 7 is a pictorial depiction of the results of expression of certainpleurocidin-like genes in different winter flounder tissues.

FIG. 8 is a pictorial depiction of the results of RTPCR of expression ofcertain pleurocidins during winter flounder development.

FIG. 9 is a pictorial depiction of the results of a study of theexpression of certain pleurocidin-like genes during winter flounderdevelopment.

FIG. 10 is a pictorial depiction of the results of a Southern analysisof certain pleurocidin genes of winter flounder.

FIG. 11 is a schematic depiction of the genomic organization of certainpleurocidin genes from winter flounder.

FIG. 12 is a schematic depiction of certain transcription factor bindingsites located upstream from pleurocidin genes from winter flounder.

FIG. 13 is a graphical depiction of results showing the impact ofpeptide NRC-15 on bacterial survival.

FIG. 14 is a graphical depiction of results showing the impact ofpeptide NRC-13 on bacterial survival.

FIG. 15 is a graphical depiction of results showing the impact ofpeptide NRC-12 on yeast survival.

FIG. 16 is a depiction of nucleotide sequences of an unspliced (A) andpartially spliced (B) cDNA encoding a type I hepcidin and a schematicdepiction of intron/exon structure of a hepcidin gene in human, mouseand salmon (C).

FIG. 17 is a depiction of certain hepcidin sequences from differentspecies shown in alignment.

FIG. 18 is a depiction of certain aligned 3′ untranslated regions ofhepcidin genes from winter flounder (A) and Atlantic salmon (B).

FIG. 19 is a pictorial depiction of the results of Southernhybridization analysis of certain hepcidins from different fish species.

FIG. 20 is a pictorial depiction of the results of an assay of theexpression of certain hepcidin and actin genes in various tissues ofwinter flounder.

FIG. 21 is a pictorial depiction of the results of an assay of theexpression of certain Type I(A) and Type 2(B) hepcidin and actin genesin various tissues of control and infected salmon.

FIG. 22 is a pictorial depiction of the results of an assay ofexpression of certain Type I(A), Type II(B) and Type III(C) hepcidin andactin genes in developing winter flounder larvae.

FIG. 23 is a schematic depiction of steps taken in an embodiment of themethod for identifying pleurocidins.

FIG. 24 is a schematic depiction of steps taken in an embodiment of themethod for identifying hepcidins.

FIG. 25 is a graphical depiction of experimental results usingantimicrobial peptide NRC-13 in the presence of 150 mM NaCe.

DETAILED DESCRIPTION OF THE INVENTION

The method of the invention builds on the surprising discovery that theflanking sequences around antimicrobial peptides, including withoutlimitation pleurocidins and hepcidins, are conserved. The method of theinvention provides a means of identifying nucleotide sequences encodingpleurocidins and hepcidins, and identifying the encoded polypeptidesequences.

In one embodiment, the method provides, generally, a way of identifyingmembers of a family of antimicrobial peptides once a single familymember has been identified. The initial family member may be an initialpeptide of interest. Initial peptides of interest can be identifiedbased on either known or reported antimicrobial activity or based onsequence similarity to other known antimicrobial peptides. Once aninitial peptide has been identified, the genomic DNA encoding it isidentified and its flanking sequences are determined.

As used herein, the term “flanking sequences” refers to nucleic acidsequences appearing at or near one or both ends of a target nucleic acidsequence encoding an antimicrobial peptide.

As used herein a nucleic acid sequence is “at or near” the end of atarget sequence if a portion of the sequence is within 50 nucleic acidsof the end of the gene (whether within the coding region or outside it).

When an initial peptide of interest is identified based on sequencesimilarity to another peptide with known antimicrobial activity, theinitial peptide preferably has an amphipathic structure and a netcharge. In some instances the charge will preferably be a net positivecharge of at least 2. In some instances, the peptide is at least 75%,85% or 95% identical in sequence to the peptide having knownantimicrobial activity. In some instances the sequence similarityidentified may relate to similarity between nucleic acid sequencesencoding the known peptide and encoding the peptide of interest. In suchinstances, the predicted peptide for the peptide of interest will beconsidered with respect to predicted charge and amphipathic structure.

For example, the prepro-sequences of pleurocidins and hepcidins tend tobe conserved. Thus, by employing nucleic acid primers specific for suchsequences, one can identify potential pleurocidin- and hepcidin-encodingsequences. Alternatively or additionally, known gene sequences of otherclasses of antimicrobial peptides can be examined to identify regionswhich appear to encode conserved prepro-sequences and a similar strategyused to identify other members of this family of peptides. Thecorresponding antimicrobial peptide encoded by such sequences can bepredicted using the general features found in most pleurocidins andhepcidins, such as, for example, a net positive charge of at least 2 andan amphipathic structure.

As used herein with respect to pre-, pro- and prepro sequences ofantimicrobial peptides, “pre” and “pro” have the following meaning:“Pre” refers to the signal peptide portion (or a functional portionthereof) of the peptide. “Pro” refers to the propiece. In pleurocidinsthe propiece is the anionic region at the carboxy terminus. In hepcidinsthe propiece is the region upstream of the mature peptide. In thenon-limiting examples disclosed herein pleurocidin primers were designedbased on the pre and pro regions, and hepcidin primers were designedbased on the pre region and the 3′ untranslated region (UTR).

PCR can be used to amplify nucleic acid sequences encoding potentialpleurocidins or hepcidins. This can be conveniently accomplished byusing a pair of PCR primers, one of which recognises a nucleic acidsequence complementary to a polynucleotide sequence encoding anamino-terminal prepro-sequence conserved in the peptide type ofinterest, and the other complementary to a 3′ conserved region in thenucleotide encoding the peptide-type of interest. It will be appreciatedthat other prepro-sequences may exist and are specifically contemplated.For example, redundancy in the genetic code allows for multiple nucleicacid sequences encoding a particular amino acid sequence. As discussedwith respect to 5′prepro-sequences, other 3′ conserved sequences mayexist and are specifically contemplated. When designing primers it isuseful to have reference to known codon usage information for thespecies in which sequence amplification is sought.

In an embodiment of the invention there is provided the use of signalsequence I or a nucleic acid sequence encoding same in identifying oramplifying potential pleurocidins. Signal Sequence I (SEQ ID NO: 305)MKFTATFL (X)_(n) (L)_(o) (F)_(p) I (F)_(q) (X)_(y) VLM (X)_(z) (V)_(r)(E)_(s) (D)_(t) (P)_(u) (L)_(v) G E (C)_(w) (G)_(x) Wherein: n is 1 to 3u is 0 or 1 o is 0 to 2 v is 0 or 1 p is 0 or 1 w is 0 or 1 r is 0 or 1s is 0 or 1 x is 0 or 1 t is 0 or 1 y is 0 or 1 z is 0 or 1 with therestriction that: x + o + p = 3, s + t = 1, u + v = 1, w + x = 1, andq + = 1.

In an embodiment of the invention there is provided the use of one orboth sequence PL1 or PL2 in identifying or amplifying potentialpleurocidins. PL1 GCCCACTTTGTATTCGCAAG (SEQ ID NO: 5) PL2CTGAAGGCTCCTTCAAGGCG (SEQ ID NO: 6)

In an embodiment of the invention there is provided the use of an acidicsequence I or a nucleic acid sequence encoding same in identifying oramplifying potential pleurocidins. Acidic Sequence I (SEQ ID NO: 306)(Y)_(a) (X)_(b) (X)_(c) (E)_(d) (X)_(e) (Q)_(f)(E)_(g) L (N/D) KR (A/S)V D (D/E) wherein: a is 0 or 1 e is 1 to 3 b is 0 or 1 f is 0 or 1 c is1 or 2 g is 0 or 1 d is 0 or 1 with the restriction that a + b = 1, c +d = 2, and e + f + g = 3.

As used in the sequences herein “X” refers to any amino acid. Nucleicacid sequences encoding signal sequence I and acidic sequence I arespecifically contemplated, as are nucleic acid sequences complementaryto such nucleic acid sequences. In an embodiment of the invention thereis provided the use of signal peptide II, III, IV, V or a nucleic acidencoding same, in the identification or amplification of hepcidins.Signal Peptide II MKXXXXAXXVXXVL (SEQ ID NO: 307) Signal Peptide IIIMKTFSVAV (SEQ ID NO: 308) Signal Peptide IV MKTFSVAVTVAVVLXFICIQQSSA(SEQ ID NO: 309) Signal Peptide V MKTFSVAVAV (T/V) (L/V) VLA (SEQ ID NO:310) (F)_(n)(V/C) (C/M) (I/F) (Q/I) X (X)_(m) S (S/T) AV P FXXV,Wherein n is 0 or 1 and m is 0 or 1.

In an embodiment of the invention there is provided the use ofprosequence I, Prosequence II or a nucleotide sequence encoding same orcomplementary to one encoding same in the identification oramplification of hepcidins. Prosequence I PEVQXLEEAXSXDNAAAEHQE (SEQ IDNO: 311) Prosequence II PFXXVX(X)_(n) (L/T) EEV (E/G) (G/S) (SEQ ID NO:312) XD (T/S) PV (A/G) XHQ,Wherein n is 0 or 1,

In an embodiment of the invention there is provided the use of HcPA3b3′and/or HcSa13′ in the identification or amplification of hepcidins.HcPa3b 3′ 3′ACAACCTCGTCCTTAGG5′ (SEQ ID NO: 313) HcSal 3′3′ACGCCCGTCCAGGAAT5′ (SEQ ID NO: 314)Non-Limiting Examples Of Uses

Antimicrobial peptides are useful in the treatment and/or prevention ofinfection in a variety of subjects, including fish, reptiles, birds,mammals, amphibians and insects.

Antimicrobial peptides are also useful for reducing bacterial growthand/or accumulation on surfaces. This is of particular benefit in thefood industry where antimicrobial peptides can be used for coatingsurfaces used in the processing, preparation, and/or packaging of food.

Antimicrobial peptides disclosed herein can be administered in a varietyof ways. In some instances, oral administration will be desirable. Sometypes of oral administration will be improved by encapsulation of thepeptides so as to allow their preferential release at a particular stagein digestion. In some instances it will be desirable to include preand/or pro sequences in the administered peptide (for example to improvestability or modulate activity). The pre and/or pro sequences can becleaved off by endogenous proteases at the appropriate stage. Peptidesmay be administered by inhalation where the subject breathes air or byaddition to water for gilled subjects. Administration by injection willin some cases be desirable. Peptides may be injected into any number ofsites. In some cases intravenous injection will be desired. In someinstances injection directly into or adjacent to the site of infectionor potential infection will be desired. In some instances topicaladministration will be desired. Where the presence of the antimicrobialpeptide is desired at a remote and specific site, or where the peptidewill be desired for a prolonged period of time, gene therapy may be usedto provide expression of one or more antibacterial peptides in thetissue(s) of concern.

Where the subject is a cultured or domesticated creature such as a fish,bird or non-human mammal, production of a transgenic variety whichexpresses one or more antibacterial peptides may be desired. Methods forproducing transgenic animals are well known. (See for example Mar.Biotechnol.4: 338,2002).

A variety of antimicrobial peptides are contemplated and fall within thescope of the invention. By way of non-limiting example, peptidescomprising the following amino acid sequences or a sequence at least 80%or 90% homologous thereto, and nucleic acid sequences encoding them arespecifically contemplated: i) GW(G/K)XXFXK (SEQ ID NO: 315) ii)GXXXXXXXHXGXXIH (SEQ ID NO: 316) iii) FKCKFCCGCCXXGVCGXCC (SEQ ID NO:317) iv) CXXCCNCC (K/H) XKGCGFCCKF (SEQ ID NO: 318) v)FKCKFCCGCRCGXXCGLCCKF (SEQ ID NO: 319) vi) XXXCXXCCNXXGCGXCCKX (SEQ IDNO: 320)

Other specific, non-limiting examples of antimicrobial sequences ofinterest can be found in Tables 4 and 11.

Antimicrobial peptides of the invention may be modified. Suchmodifications may in some instances improve the peptides′ stability oractivity. Examples of modifications specifically contemplated include:

-   -   conservative amino acid substitutions (acidic with acidic, basic        with basic, neutral with neutral, polar with polar, hydrophobic        with hydrophobic, etc.)    -   addition of positively charged amino acids (lysine, arginine,        histidine) at either or both ends    -   replacement of amino acids with others unlikely to result in        structural changes including D-amino acids and peptidomimetics    -   deletion of one or more amino acids    -   modifications at C-terminal or N-terminal ends, including methyl        esters and amidates    -   cyclised versions of the peptides (which may result in increased        stability without adversely affecting activity)

EXAMPLES Methods

Fish Rearing

Winter flounder larvae were reared as described (Douglas, Gawlicka etal. 1999), the disclosure of which is incorporated herein by reference.Saint John River stock Atlantic salmon (Salmo salar L.) were maintainedin single-pass, heated, dechlorinated fresh water at 12° C. in theDalhousie University Aquatron facility in Halifax, Nova Scotia. All fishwere euthanised with an overdose of tricaine methanesulfonate (MS 222,0.1 g L⁻¹, Argent Chemical Laboratories, Inc., Redmond, Wash., USA)prior to sampling. All animal procedures were approved by the DalhousieUniversity Committee for Laboratory Animals and the National ResearchCouncil—Halifax Local Animal Care Committee.

Bacterial Challenge

Aeromonas salmonicida subsp salmonicida strain A449 (Trust et al. 1983)was cultured to mid-logarithmic growth in Tryptic Soy Broth (TSB) at 17°C. The absorbance at 600 nm of the bacterial suspension was determinedand the bacteria were resuspended to approximately 5×10⁷ cfu mL⁻¹ insterile Hanks Balanced Salt Solution (HBSS). Three salmon (200 g each)were anaesthetised with 50 mg L⁻¹ TMS, injected intraperitoneally with2.5×10⁶ cfu bacteria in 50 μL HBSS and allowed to recover in freshwater. Uninjected fish from the same cohort were maintained in separatetanks as controls. Three days post-injection, control and infectedsalmon were euthanised as described above and samples of tissuesremoved. Blood was drawn from the caudal vein into a heparinisedcontainer. To confirm that the fish were positive for A. salmonicida,the posterior kidney of both infected and control fish were swabbed andused to inoculate tryptic soy agar (TSA) that was incubated at roomtemperature overnight. Atlantic halibut tissue samples were obtainedfrom a bacterial challenge study performed at Bedford Institute ofOceanography, Dartmouth, Nova Scotia.

Sampling

Tissues (oesophagus, stomach, pyloric caecae, liver, spleen, intestine,anterior kidney, posterior kidney, gill, skin, ovary, rectum, heart,muscle and brain) were removed into RNALater (Ambion, Austin, Tex., USA)and kept at −80° C. until used. Samples of winter flounder larvae atdifferent stages and juveniles were rinsed in RNALater (Ambion, Austin,Tex., USA), transferred into 1.5 ml Eppendorf tubes containing 0.5-1.25ml RNALater, and kept at −80° C. until used.

Pleurocidins

The general approach followed is shown in FIG. 24

Isolation of Pleurocidin cDNA

A cDNA library constructed from winter flounder skin (Gong et al 1996)was screened using degenerate oligonucleotides (PleuroA, PleuroB; Table1). The library was plated at 80,000 phage/plate and duplicate lifts toHyBond filters were made of each of eight plates. A mixture ofradioactively end-labelled PleuroA and PleuroB probes was hybridisedwith the filters at 50° C. using standard procedures, and the filterswere washed in 1×SSC/0.1% SDS at 50° C. for 45 min. Plaques that showedmatching hybridization signals on both duplicate filters were picked andthe library rescreened until 100% purity of the recombinant plaques wasobtained. Two recombinants were completely sequenced using an ABI373stretch automated sequencer and the AmpliTaqFS Dye Terminator CycleSequencing Ready Reaction kit (Perkin-Elmer, Foster City, Calif., USA).Sequence data were analyzed using Sequencher (Gene Codes, Inc., AnnArbor, Mich., USA) and DNA Strider. The amino-terminal signal sequencewas predicted using SignalP (http://www.cbs.dtu.dk/services/SignalP).The Helical Wheel routine of the GCG package (http://www.gcg.com) wasused to model the helical structure of the predicted antimicrobialpeptide sequences.

Genomic PCR

Genomic sequences were amplified using two sets of primers specific tothe winter flounder pleurocidin cDNA (PL1/PL2 and PL5′/PL3′; Table 1;FIG. 1). The amplification conditions were: 1 min at 94° C.; 35 cyclesof 30 s at 940 C; 30 s at 52° C., 90 s at 72° C.; and 2 min at 72° C.,and products were resolved on a 1% agarose gel. Bands were excised fromthe gel, extracted using Gene-Clean (Bio101, La Jolla, Calif., USA) andcloned into the Topo TA2.1 vector (Invitrogen, Carlsbad, Calif., USA) asrecommended by the manufacturers. Several isolates from eachtransformation were sequenced and analyzed as described above. Intronpositions were identified by comparison with the cDNA sequence.

Identification of Additional Winter Flounder Pleurocidin-Like Sequencesby RT-PCR

Total RNA was isolated from winter flounder skin and intestinesubstantially as described in Douglas, Gawlicka et al (1999). Reversetranscription of 2 μg of total RNA was performed using the RETROScriptkit (Ambion, Austin, Tex., USA) according to the manufacturer'srecommendation. PCR was performed using PL3′ and a primer correspondingto the amino terminus of the precursor polypeptide (PL5′; Table 1). Theamplification conditions were: 1 min at 94° C.; 32 cycles of 30 s at 94°C., 30 s at 50° C., 90 s at 72° C.; and 2 min at 72° C. and productswere resolved on a 2% NuSeive gel. Bands were excised, cloned andsequenced as described above.

Identification of Additional Pleurocidin-Like Sequences From DifferentTissues

Tissue-specific expression of pleurocidin was investigated by northernanalysis using polyadenylated RNA (500 ng) from adult skin, liver,ovary, muscle, spleen, pyloric caeca, stomach and intestine. The entireinsert from the cDNA clone corresponding to WF2 was radioactivelylabelled and incubated with the blot overnight at 60° C. in UltraHybhybridisation solution (Ambion, Austin, Tex., USA). The blot was washedto a stringency of 50° C. in 1×SSC/0.1% SDS for 1 h before exposure toX-ray film. RT-PCR was also employed using primers specific to WF1,WF1a, WF2, WF3, WF4, WFYT and WFX (Table 2) to assay expression of thedifferent pleurocidin-like variants in various tissues. The conditionsused were as described in the preceding paragraph except that theannealing temperature was 52° C.

Identification of Additional Pleurocidin-Like Sequences From DifferentDevelopmental Stages

Two larval time series were used to assess developmental expression ofpleurocidin-like genes. In the first, RNA was isolated from pooledsamples of twenty whole larvae (5 and 13 dph), ten whole metamorphosinglarvae (20 dph) and newly metamorphosed larvae (27 dph), gut tissue oftwo juveniles (41 dph), skin from the upper and lower side of adult fishand tissue from adult upper and lower intestine. RNA was isolated asdescribed (Douglas, Gawlicka et al. 1999), the disclosure of which isincorporated herein by reference, and the assays were performed usingthe primers PL5′ and PL2 and conditions described above for RT-PCR.Amplification of the actin mRNA was performed as previously described(Douglas, Bullerwell et al. 1999), the disclosure of which isincorporated herein by reference, to confirm the steady level ofexpression of a housekeeping gene and to provide an internal control forpleurocidin expression. In the second larval time series, RNA wasisolated from pooled samples of twenty whole larvae (hatch, 5 and 9dph), ten whole larvae (15, 20, 25, 30 and 36 dph) and gut tissue of twojuveniles (41 dph). Assays were performed using primers specific to WF1,WF1a, WF2, WF3, WF4, WFYT and WFX (Table 2) to determine expression ofthe different pleurocidin-like variants at different stages ofdevelopment. The conditions used were as described in the precedingparagraph.

Southern Analysis

Southern analysis of BamHI- and SstI-digested genomic DNA from winterflounder, three other flatfish (American plaice Hippoglossoidesplatessoides Fabricius, Atlantic halibut Hippoglossus hippoglossus L.and yellowtail flounder Pleuronectes ferruginea Storer), haddock(Melanogrammus aeglefinus L.), pollock (Pollachius virens L.) and smelt(Osmerus mordax Mitchill) was performed sequentially using the entireinserts from genomic clones corresponding to WF1, WF2, WF3 and WF4 asprobes. Hybridisations were performed overnight at 65° C. as previouslydescribed (Douglas, Gallant et al. 1998), the disclosure of which isincorporated herein by reference, and the blots were washed at 65° C. in0.5×SSC/0.1% SDS for 1 h and exposed to X-ray film. Blots were strippedby incubating twice in boiling 0.5% SDS and checked for residual signalby exposure to X-ray film overnight.

Identification of Additional Pleurocidin-Like Sequences From Other FishSpecies

Total RNA was isolated from skin and intestine of yellowtail flounder,witch flounder and Atlantic halibut and reverse-transcribed as describedabove (RT-PCR analysis). Total genomic DNA was isolated from milt ofyellowtail flounder, witch flounder, American plaice, Atlantic halibutand tissue samples of Petrale sole, C—O sole, English sole, Starryflounder, European plaice, Greenland halibut and Pacific halibut. Twosets of primers specific to the winter flounder pleurocidin cDNA(PL1/PL2 and PL5′/PL3′; Table 1; FIG. 1) were used and the amplificationconditions were: 1 min at 94° C., 32 cycles of 30 s at 94° C.; 30 s at50° C., 90 s at 2 min at 72° C. Products were resolved on a 2% NuSeivegel, bands excised, cloned and sequenced as described above.

FIG. 1 is a textual and graphical depiction of WF2 pleurocidin fromwinter flounder A. Nucleotide sequence of cDNA for pleurocidin fromwinter flounder isolated from the skin library. The positions of primersused for PCR are underlined and the deduced amino acid sequence is shownin upper case letters below the nucleotide sequence. Arrows indicate themature 5′ and 3′ termini of the pleurocidin peptide and diamondsindicate the positions of introns. The single SstI restrictionendonuclease site (GAGCTC) and the putative polyadenylation site(aataaa) are indicated in boldface. B. Hydrophobicity plot of predictedpleurocidin polypeptide WF2 constructed using the Kyte-Doolittle optionof DNA Strider (Marck 1992). The borders of the mature pleurocidin areindicated by vertical arrows. C. Diagrammatic representation of helicalstructure of predicted pleurocidin polypeptide WF2 constructed using theHelical Wheel routine of GCG. Hydrophobic residues and glycines areboxed and polar residues are not. The first amino acid (G) of the maturepolypeptide is found at the top of the wheel.

Identification of Pleurocidin-Like Sequences in the Winter FlounderGenome

A winter flounder genomic λ-GEM library was screened using aradioactively labeled probe for pleurocidin (WF2; Douglas et al., 2001).Four clones were picked and replated until 100% purity was achieved. Theclones were mapped using BamHI, SstI, XhoI and Eco RI and two clones(λ1.1 and λ5.1) that differed in restriction pattern were selected forsequencing. Both clones were completely sequenced using an ABI373stretch automated sequencer and the AmpliTaqFS Dye Terminator CycleSequencing Ready Reaction kit (Perkin Elmer, Foster City, Calif., USA.Transcription factor binding sites were identified using WWW Signal Scan(http://bimas.dcrt.nih.gov/molbio/signal/) with the TransFac and TFDdatabases and promoters were detected using the eukaryotic promoterprediction by neural network software available at the Baylor College ofMedicine(http://searchlauncher.bcm.tmc.edu/seq-search/gene-search.html).

Hepcidins

The general approach followed is depicted in FIG. 24

Molecular Characterisation of Hepcidin cDNAs

Eight ESTs showing high similarity to human hepcidin were identifiedfrom the winter flounder EST database (Douglas, Gallant et al. 1999) andfour from the Atlantic salmon database (Douglas, Tsoi et al. 2002).Using these sequences to screen dbEST, BLASTX analysis revealed tworelated sequences from Japanese flounder (C23298.1 and C23432.1), onesequence from rainbow trout (AF281354_(—1)) and five identical sequencesfrom medaka (AU178966, AU179222, AU179314, AU179768 and AU180044).Sequence data were analyzed using Sequencher (Gene Codes, Inc., AnnArbor, Mich., USA) and DNA Strider (Marck 1992). Alignments andsimilarity matrices were calculated using ClustalW (Thompson, Higgins etal. 1994) and graphically visualised using SeqVu (Garvan 1996). Theon-line servers PSORT (http://PSORT.nibb.ac.ip), Compute pI(http://expasy.hcuge.ch/cgi-bin/pi tool), and Network Protein Sequence@nalysis (http://npsa-pbil.ibcp.fr/cpi-bin/secpred_consensus.pl) wereused to predict N-terminal signal sequences, pI and secondary structure,respectively. The secondary structure prediction program utilized sevendifferent algorithms (for details, see web site) and provided aconsensus prediction based on these results.

Southern Hybridisation

Total genomic DNA was prepared from winter flounder (Pleuronectesamericanus), yellowtail flounder (Pleuronectes ferruginea), witchflounder (Glyptocephalus cynoglossus), Japanese flounder (Paralichthysolivaceus), American plaice (Hippoglossoides platessoides), Atlanticsalmon (Salmo salar), haddock (Melanogrammus aeglefinus), smelt (Osmerusmordax), hagfish (Eptatretus burgeri), tiger shark (Scyliorhinustorazame) and white sturgeon (Acipenser transmontanus) as previouslydescribed (Douglas, Bullerwell et al. 1999), the disclosure of which isincorporated herein by reference. DNA (7.5 □g) was digested with SstIaccording to the manufacturer's recommendations and the fragmentsresolved on a 1% agarose gel. A 104 bp probe corresponding to amino acidresidues WMENPT . . . GCGFCC (SEQ ID NO: 321 and 322 respectively) ofType I winter flounder hepcidin was labeled using the DIG Labelling Kit(Roche Applied Science, Laval, PQ, Canada) and hybridized to themembrane for 2 h at 42° C. using the Easy Hyb kit (Roche AppliedScience, Laval, PQ, Canada). The membrane was washed in 0.2×SSC at 65°C. and signal detected using the DIG Luminescent Detection Kit (RocheApplied Science, Laval, PQ, Canada).

Identification of Additional Hepcidin-Like Sequences by RT-PCR

Primers were designed based on the cDNA sequences determined in thisstudy (Table 3). Amplification of actin mRNA was performed to confirmthe steady-state level of expression of a housekeeping gene and providean internal control for the hepcidin gene expression analyses. Controlswere performed using single primers to eliminate single primer artifactsand without reverse transcription to eliminate amplification productsarising from contaminating genomic DNA.

Total RNA was isolated from tissues of uninfected adult winter flounderand uninfected and infected adult salmon and halibut using the RNAWizKit (Ambion, Austin, Tex., USA) according to the manufacturer'srecommendations. Tissues were homogenized using a 7mm generator on aPolytron standard rotor stator homogenizer (Kinematica). In addition,RNA was isolated from pooled samples of twenty whole larvae (hatch, 5and 9 dph), ten whole larvae (15, 20, 25, 30 and 36 dph), gut tissue oftwo juveniles (41 dph) and adult winter flounder liver. To eliminatecontaminating DNA, the Ambion DNA-free TM protocol was used as directed.Briefly, 4 units of DNase 1 was added to the resuspended RNA andincubated for 1 hour at 37 C. After incubation, DNAse InactivationReagent was added to remove the enzyme and RNA concentrations weredetermined using a Beckman DU-64 Spectrophotometer.

First strand cDNA was synthesized from 1 μg of total RNA using theRetroScript kit (Ambion, Austin, Tex., USA) and aliquots of the reactionproducts were subjected to PCR using rTaq polymerase (Amersham PharmaciaBiotech AB, Uppsala, Sweden) or the Advantage2 PCR kit (Clontech, PaloAlto, Calif., USA). The primers and annealing temperatures are listed inTable 3. The amplification conditions were: 1 min at 95° C.; 32 cyclesof 15 s at 95° C.; 30 s at the annealing temperature, 30 s at 68° C.;hold at 4° C. Amplification products were resolved on a 2% NuSieveagarose gel with a 100 bp ladder as a marker (Gibco BRL, Gaithersburg,Md., USA) and the amount of each product was quantified using a GelDoc1000 video gel documentation system (BioRad, Mississauga, Ont., Canada)with the Multianalyst software.

Identification of Additional Hepcidin-Like Sequences From Other FishSpecies

Total RNA was isolated from liver and spleen of bacterially challengedAtlantic halibut and Atlantic salmon and reverse-transcribed asdescribed above (RT-PCR analysis). Two sets of primers were used (seelegend, FIG. 2) and the amplification conditions were: 2 min at 94° C.;32 cycles of 30 s at 94° C.; 30 s at 52° C., 30 s at 72° C.; and 2 minat 72° C. Products were resolved on a 2% NuSeive gel, bands excised,cloned and sequenced as described above.

Bacterial Strains and Candida albicans

All strains used in this study are listed in Table 5. Most non-fishbacterial strains as well as Candida albicans were grown at 37° C. inMueller-Hinton Broth (MHB; Difco Laboratories, Detroit), while the fishbacteria were maintained at 16° C. in Tryptic Soy Broth (TSB; Difco,5g/l NaCl). All strains were stored at −70° C. until they were thawedfor use and sub-cultured daily. The following strains, Pseudomonasaeruginosa K799 (parent of Z61), Pseudomonas aeruginosa Z61 (antibioticsupersusceptible), Salmonella typhimurium 14028s (parent of MS7953s),Salmonella typhimurium MS7953s (defensin supersusceptible), as well asStaphylococcus epidermidis (human clinical isolates) andmethicillin-resistant Staphylococcus aureus (MRSA; isolated by Dr. A.Chow, University of British Columbia) have been kindly donated by ProfR. E. W. Hancock, University of British Columbia.

Escherichia coli strain CGSC 4908 (his-67, thyA43, pyr-37), auxotrophicfor thymidine, uridine, and L-histidine (Cohen et al., 1963) was kindlysupplied, free of charge, by the E.coli Genetic Stock Centre (YaleUniversity, New Haven, Conn.). MHB supplemented with 5 mg/L thymidine,10 mg/L uridine and 20 mg/L L-histidine (Sigma Chemical Co., St. Louis,Mo.), was used to grow E.coli CGSC 4908 unless otherwise specified.

Two field isolates of the salmonid pathogen Aeromonas salmonicida arefrom the IMB strain collection.

Minimum Inhibitory Concentrations

The activities of the antimicrobial peptides were determined as minimalinhibitory concentrations (MICs) using the microtitre broth dilutionmethod of Amsterdam (Amsterdam, 1996), as modified by Wu and Hancock(1999). Serial dilutions of the peptide were made in water in 96-wellpolypropylene (Costar, Coming Incorporated, Coming, New York) microtiterplates. Bacteria or C. albicans were grown overnight to mid-logarithmicphase as described above, and diluted to give a final inoculum size of10⁶ cfu/ml. A suspension of bacteria or yeast was added to each well ofa 96 well plate and incubated overnight at the appropriate temperature.In the case of E. coli CGSC 4908, supplemented MHB was used. Inhibitionwas defined as growth lesser or equal to one-half of the growth observedin control wells, where no peptide was added. Three repeats of each MICdetermination were performed.

Killing Assays

Survival of bacteria and C. albicans upon exposure to selected peptidesapplied at their minimal inhibitory concentrations (MICs) and ten timestheir MICs was measured using standard methodology. The test organismswere grown in MHB and exposed to the peptides. At the specified timeintervals equal aliquots were removed from the cultures, plated on MHBplates, and the resulting colonies were counted. Percentage survival wasplotted against time on a logarithmic scale. Two repeats of eachexperiment were performed.

Preparation of a Synthetic Antimicrobial Peptide

Prediction of active cationic peptide sequences.

The mature peptide sequences from FIG. 3 (pleurocidin-like peptidesequences deduced from nucleotide sequences of genes and PCR productsamplified from fish tissues) constituted the basis of sequenceselection.

Upon extensive sequence analysis, sequences were selected for peptidesthat possessed a net positive charge and had their hydrophilic andhydrophobic residues well separated spatially in models.

Also, generally those peptide genes that were likely to be expressed(possessed promoters, were transcribed, etc.) were produced, althoughpseudogenes were also included in the panel.

The exact start/end residues were decided upon based on several factors:

a) In most cases the N-terminus of the mature peptide was well-defined,since it followed directly the conserved signal peptide region, andaligned well with other mature peptides.

b) Wherever a straightforward determination on the N-terminal amino acidwas not possible, an attempt was made to preserve GW or GF at theN-terminus, as this is frequently encountered among cationic peptides.

c) In addition, two versions of WF1a (NRC-2 and NRC-3) were produced:one contained N-terminal GRRKRK (SEQ ID NO: 323), and the other did not;this was done because it was hypothesized that the presence of thehighly positively charged GRRKRK (SEQ ID NO: 323) would improveactivity.

d) Although in some cases the C-terminus of the mature peptide was alsowell defined, since it was followed directly by a conserved acidicpropiece, significant ambiguity as to the C-terminal amino acid existedamong many peptides. Generally, two rules were followed in deciding uponC-terminal amino acids:

1. wherever glycine appeared at or near the C-terminus, it wasconsidered to be a precursor for carboxy-terminus amidation;

2. large numbers of negatively charged amino acids near the C-terminuswere generally considered to be a part of the propiece and not matureactive peptide and were not included in the sequence.

In order to estimate the net charge, K and R were assumed to have thevalue of +1, H of +1/2, D and E of −1, and C-terminal amidation wascounted as an additional +1. The EMBOSS Pepwheel and Pepnet internettools available through an NRC mirror site(http://bioinfo.pbi.nrc.ca:8090/EMBOSS/index.html) were used to analysethe separation of hydrophilic and hydrophobic residues in helical wheeland helical net models.

All antimicrobial peptides used in this study were synthesized byN-(9-fluorenyl) methoxy carbonyl (Fmoc) chemistry at the Nucleic AcidProtein Service (NAPS) unit at the University of British Columbia.Peptide sequences are shown in Table 4. Peptide purity was confirmed byHPLC and mass spectrometry analysis in each case. In the case of NRC-7further purification by RP-HPLC was performed until homogeneity of thesample was obtained.

Peptides produced according to the above steps are screened forantimicrobial activity in vitro by standard means. Those peptidesshowing in vitro antimicrobial activity are useful as antimicrobialpeptides for use in vivo and for the treatment of surface, etc.

EXAMPLES Results

Pleurocidins

cDNA sequence

The two clones isolated from the winter flounder skin cDNA library wereidentical in sequence to each other and to the genomic PCR product WF2after introns were removed (see below). They contain 356 bp and encodean open reading frame of 68 amino acids (FIG. 1A). There is a5′-untranslated region of 26 bp and a 3′-untranslated region of 84 bp,excluding the polyA tail. A canonical polyadenylation signal AATAAA isfound 22 bp upstream of the polyA tail. The first 22 amino acids of theopen reading frame form a highly hydrophobic domain (FIG. 1B) predictedto be a signal peptide with a cleavage site that precisely matches theamino terminus of the mature pleurocidin. The predicted amino acidsequence of residues 23-47 exactly matches the published amino acidsequence of mature pleurocidin (arrows, FIG. 1A). The mature peptide canassume an amphipathic helix that contains a predominance of positivelycharged amino acids on one face and hydrophobic amino acids on the other(FIG. 1C). The carboxy-terminal 21 amino acids form a negatively chargeddomain that is not present in the mature pleurocidin, confirming therecent report of Cole et al. (2000).

Genomic PCR

Four distinct bands (WF1-4) were amplified using primers PL5′ and PL3′(FIG. 4). Sequence analysis of each product was consistent with thesizes of the bands and verified that each amplification product wasdifferent (Table 6). Two distinct bands were amplified using primers PL1and PL2 that corresponded to WF2 and WF4 containing additional upstreamand downstream sequence (data not shown). When the intron sequences wereremoved, the sequence of WF2 exactly matched that of the pleurocidincDNA clone isolated from the skin library (FIG. 1A).

FIG. 4 is a depiction of the results of PCR amplification ofpleurocidin-like sequences from winter flounder genomic DNA.Amplification products (P) were resolved on a 1% agarose gel using the100 bp ladder as molecular weight markers (M). Products visible asdistinct bands are labeled WF1 (00 bp), WF2 (810 bp), WF3 (650 bp) andWF4 (510 bp).

All four of the pleurocidin-like genes contained two introns within thecoding sequence and three of the genes showed identical intron locations(WF1, WF2 and WF4). However, the position of the second intron in WF3occurred upstream of those of the other genes, resulting in a shortersecond exon and longer third exon. The sizes and sequences of theintrons varied among the four pleurocidin genes (Table 6). Evidence fromthe two more extensive genomic sequences of WF2 and WF4 obtained usingprimers PL1 and PL2 indicates that a third intron immediately upstreamof the initiation codon is also a feature of this gene family (FIG. 5).This was also noted for the genomic sequence reported by Cole et al(Cole, Darouiche et al. 2000).

An alignment of the predicted amino acid sequences is shown in FIG. 6.The positions of the introns (indicated by vertical arrows) weredetermined by comparison with the corresponding RT-PCR and cDNA-derivedsequences. The positions of the mature peptide were determined bycomparison with the published amino acid sequence of pleurocidin (Cole,Weis et al. 1997). All of the predicted mature polypeptides could assumeamphipathic α-helical structures similar to that shown in FIG. 1C,although the positively charged portions were not as striking in WF1 andWF3 as in WF2 and WF4 (data not shown).

FIG. 5 describes extended genomic sequence of WF4 obtained by PCR usingprimers PL1/PL2. Introns are indicated in lower case and coding sequencein upper case The positions of the primers PL1 and PL2 used for PCR areunderlined.

FIG. 6 describes Alignment of predicted polypeptide sequences of fivewinter flounder pleurocidin family members. Large vertical arrowsindicate the positions where introns were found in the genomicsequences. The second intron of WF3, indicated by a small verticalarrow, is found more upstream than those of the other genes. Thepredicted polypeptide sequences of dermaseptin B1 (Amiche et al. 1994)and ceratotoxin B (Marchini et al. 1995) are shown below the pleurocidinfamily members. Boxed amino acids are shared by half of the sequences.

Identification of Additional Pleurocidin-Like Sequences From DifferentTissues

Northern analysis was only able to detect pleurocidin transcripts inskin (data not shown). However, the more sensitive RT-PCR assayindicated that pleurocidin was also expressed in other tissues,particularly gill and gut. Using primers PL5′ and PL3′, two bards wereobtained from winter flounder skin (265 and 175 bp) and two fromintestine (215 and 175 bp). Sequence analysis of several clones of eachsize showed that the 265 bp winter flounder skin clones corresponded tothe genomic sequence of WF1 when intron sequences were removed (Table7). Five of the 175 bp clones from skin and two of the 175 bp clonesfrom intestine corresponded to the genomic sequence of WF2. This isconsistent with results of northern analysis using the cDNA clonecorresponding to the WF2 probe that showed hybridisation only to200-nucleotide mRNA from the skin (data not shown). On the other hand,nine of the 175 bp clones from intestine and four of the 175 bp clonesfrom skin corresponded to the genomic sequence of WF3. No RT-PCRproducts were obtained that corresponded to WF4. All seven of the 215 bpintestine clones corresponded to a novel family member (WF1a) notrepresented by any of the winter flounder genomic sequences determinedin this study.

Using primers specific to each of the pleurocidin-like variants reportedabove, as well as to additional pleurocidin-like variants identified onLambda clones, we were able to demonstrate that different variants wereexpressed in different tissues (FIG. 7). WF2, WF3 and WFYT showed theexpression in the widest distribution of tissues, whereas WF1 and WF4were expressed in mainly in the gill and skin, and WFX was onlyexpressed in the skin. Transcripts of WF 1 a could not be detected inany tissue.

FIG. 7 describes the expression of specific pleurocidin-like genes indifferent tissues of winter flounder. Tissues were esophagus (E),pyloric stomach (PS), cardiac stomach (CS), pyloric caeca (PC), liver(L), spleen (SP), intestine (I), rectum (R), gill (G), brain (B) andskin (SK). Markers (M) were the 100 bp ladder. Primers were specific toeach pleurocidin variant (Table 2)

Identification of Additional Pleurocidin-Like Sequences From DifferentDevelopmental Stages

Using primers PL5′ and PL2 (Table 1) from highly conserved regions ofthe pleurocidin-like peptides, low levels of transcripts were evident at5 dph and increased during development (FIG. 8). Strong signals wereobtained from adult skin and weak signals from intestinal tissue.Expression of the housekeeping gene, actin, was relatively constantthroughout development.

Using primers specific to each of the pleurocidin-like variants reportedabove, as well as to additional pleurocidin-like variants identified onLambda clones, it was demonstrated that different variants wereexpressed at different times during development (FIG. 9). WFXtranscripts were only detectable at 20 dph, and WF2, WF3 and WFYT weredetectable in premetamorphic larvae and metamorphic juveniles. Noexpression of WF1 and WF4 was detectable at any stage of development.

FIG. 8 describes Reverse transcription-polymerase chain reaction assayof pleurocidin expression. Samples are from larvae (5 and 13 dph),metamorphosing larvae (20 dph), newly metamorphosed larvae (27 dph),juveniles (41 dph), skin from the lower (LS) and upper side (US) of thefish and tissue from the lower (LI) and upper (UI) intestine. Primersspecific for pleurocidin (panel A) and actin (panel B) were used.

FIG. 9 describes Expression of specific pleurocidin-like genes duringwinter flounder larval development. Samples are from larvae (5, 9 and 15dph), metamorphosing larvae (20 dph), newly metamorphosed larvae (25, 30and 36 dph) and juveniles (41 dph). Controls using the 5′ or 3′ primersalone and with no template (NT) are also shown. Primers were specific toeach pleurocidin variant (Table 2).

Southern Analysis

Positive signals were specific to flatfish DNA using the WF1, WF2, WF3and WF4 genomic probes (FIG. 10). No signals were detected with haddock,pollock or smelt DNA (data not shown). All four probes showedhybridisation to common SstI and BamHI bands from the DNAs of all fourflatfish, indicating that the genes are clustered on these genomes. Thesizes of the hybridising fragments from the winter flounder digest aregiven in Table 8.

FIG. 10 describes Southern analysis of pleurocidin genes of winterflounder (WF), yellowtail flounder (YF), American plaice (AP) andAtlantic halibut (AH). Total genomic DNA (7.5 μg) was digested withBamHI (B) or SstI (S) and the fragments resolved on a 1.0% agarose gel.The blot was hybridized successively with probes corresponding to WF1,WF2, WF3, and WF4. Markers (M) are lambda DNA digested with StyI (24.0,7.7, 6.2, 3.4, 2.7, 1.9, 1.4, 0.9 Kb).

Identification of Additional Pleurocidin-Like Sequences From Other FishSpecies

An alignment of the deduced amino acid sequences of pleurocidin-likepeptides from American plaice, yellowtail flounder, witch flounder andAtlantic halibut is shown in FIG. 3. Sequences were obtained fromgenomic DNA of Petrale sole, C—O sole, English sole, starry flounder,European plaice, Greenland halibut and Pacific halibut. Highconservation is present in the signal peptide and acidic propieceregions, whereas the portion corresponding to the mature peptide showsmuch more variability.

FIG. 3 describes Alignment of pleurocidin-like peptide sequences deducedfrom nucleotide sequences of genes and PCR products amplified from skinand/or intestine of the following species: winter flounder (WF),yellowtail flounder (YF), witch flounder (GC), American plaice (AP) andAtlantic halibut (AH). Specific non-limiting examples ofpleurocidin-like sequences identified are shown in Table 4. Non-limitingexamples of cDNA and/or genomic sequences are provided in Appendix I.

Identification of Pleurocidin-Like Sequences in the Winter FlounderGenome

Two clones containing fragments of 12.5 and 15.6 kb, respectively, wereisolated from a genomic library from winter flounder. The 12.5 kbfragment encoded the gene corresponding to WF2 and two pseudogenes. The15.6 kb fragment encoded the gene corresponding to WF1, one pseudogeneand two previously undescribed pleurocidin-like sequences referred to asWFX and WFYT. A schematic of the gene arrangement is shown in FIG. 11.Scanning of the sequences upstream of the coding sequence revealed acanonical eukaryotic promoter, TATA and CAAT boxes as well as highlyconserved sites for several transcriptions factors including NF-IL6, APIand α-interferon (FIG. 12). No promoter sequences were identifiedupstream of pseuodgenes.

FIG. 12 describes Locations of transcription factor binding sitesupstream of pleurocidin genes and pseudogenes. Promoters are indicatedby hatched boxes, introns by solid boxes and genes and exons by stippledboxes.

Prediction and Assessment of Antimicrobially Active Peptide Sequences

The minimal inhibitory concentrations of the chemically producedpeptides against a wide range of baterial pathogens and C. albicans weredetermined and are shown in Table 9. Generally speaking many peptidesshowed the ability to inhibit the growth of a broad spectrum ofbacterial pathogens and C. albicans. Particularly good examples ofpeptides with a broad spectrum of antimicrobial activity are the threepeptides derived from American plaice (NRC-11, NRC-12, and NRC-13) andthree peptides derived from witch flounder (NRC-15, NRC-16, and NRC-17).Of those, NRC-15, NRC-13, and NRC-12 showed ability to killmethicillin-resistant S. aureus (FIG. 13), P. aeruginosa (FIG. 14) andC. albicans (FIG. 15), respectively.

FIG. 13 describes Survival of a Gram-positive bacterium(methicillin-resistant Staphylococcus aureus—MRSA) upon exposure toNRC-15 at its minimal inhibitory concentration (MIC) and ten times itsMIC. S. aureus was grown in Mueller-Hinton broth and exposed to NRC-15at its MIC and ten times its MIC. At the specified intervals equalaliquots were removed from the culture, plated on MHB plates, and theresulting colonies were counted.

FIG. 14 describes Survival of a Gram-negative bacterium (Pseudomonasaeruginosa) upon exposure to NRC-13 at its minimal inhibitoryconcentration (MIC) and ten times its MIC. P. aeruginosa was grown inMueller-Hinton broth and exposed to NRC-13 at its MIC and ten times itsMIC. At the specified intervals equal aliquots were removed from theculture, plated on MHB plates, and the resulting colonies were counted.

FIG. 15 describes Survival of a yeast (Candida albicans) upon exposureto NRC-12 at its minimal inhibitory concentration (MIC) and ten timesits MIC. C. albicans was grown in Mueller-Hinton broth and exposed toNRC-12 at its MIC and ten times its MIC. At the specified intervalsequal aliquots were removed from the culture, plated on MHB plates, andthe resulting colonies were counted.

In addition to demonstrating that pleurocidin-like peptides are activeagainst a wide range of bacteria as well as C. albicans, the resultsindicate which factors should preferably be considered in selectingantimicrobially active peptides from genomic sequences.

Firstly, a notable group of peptides with poor or no observed activitieswere peptides derived from pseudogenes (NRC-8, NRC-9, NRC-10). Theseresults indicate that peptides capable of being expressed in the hostorganism may be better candidates for antimicrobials.

Secondly, the previously described N-terminal GRRKRK in WF1a (FIG. 2)proved to be a determinant of antimicrobial activity in NRC-3 as shownby the fact NRC-2 (identical to NRC-3 but missing the aforementionedfragment) was only marginally active (Table 9). This result stresses theimportance of carefully selecting the start/end residues in the maturepeptide, wherever these are not apparent in the originalpre-pro-sequence.

Thus in an embodiment of the invention there is provided a group ofpleurocidin-related antimicrobial peptides having the amino acidsequence GRRKRK. It will be appreciated that pleurocidin-likeantimicrobial peptides lacking this sequence also exist and arespecifically contemplated herein.

The previously described principles of: selecting positively chargedpeptides with good separation of hydrophilic and hydrophobic residues inhelical wheel models, preserving GW or GF at the N-terminus, amidatingthe C-terminus where glycine was present, and cropping off clusters ofacidic C-terminal amino acids were successful in selectingantimicrobially active peptides.

Peptides of the invention can be used at a range of pH's, saltconcentrations, and temperatures. These peptides are useful againstpathogens grown in biofilms or under any other conditions for pathogengrowth or culture. See for example FIG. 25 in which the ability ofNRC-13 to kill P. aeruginosa K799 in 50 mM NaCl is shown. NRC-13 wasadded to a culture of P. aeruginosa supplemented with 150 mM NaCl to afinal concentration of 4 μg/ml (□) or 40 μg/ml (Δ), representing the MICand 10×MIC, respectively. A control with no peptide added is also shown(♦).

Peptides may be used alone or in combination with one or both of theirpre-and pro-sequences.

Peptides of the invention have many uses, including as antibacterial,antifungal, antiviral, anti-cancer, and antiparasitic agents, includingin combination with other antibiotics, anti-infectives, andchemotherapeutants as well as with each other.

Peptides can be used as immunomodulatory agents such as for woundhealing, tissue regeneration, anti-sepsis, immune promoters, etc.including in combination with other agents.

The peptides can be delivered topically (including e.g.,aerosols-especially for respiratory tract infections in CF patients,ointments, lotions, rinses, eyewashes, etc.), systemically (includinge.g. IV, IP, IM, subcutaneously, intracavity or transdermally) and,orally (e.g. pills, liquid medication, capsules, etc.).

Delivery via encapsulation, including in liposomes, proteinoids iscontemplated, as is delivery in transgenic systems involvingagricultural animals and/or plants.

Peptides can be used as protective coatings on medical devices(including catheters, etc, food preparation machinery and packaging.

Examples of antibiotics which can be used together with peptidesdisclosed herein in aquaculture operations include: Terramycin Aqua(oxytetracycline), Romet, (sulfadimethoxine and ormetroprim), andTribrissen (trimethoprim and sulfadiazine. In the hatchery, dipping informaldehyde can be used together with peptides disclosed herein.Peptides can be used in combination with each other and/or incombination with conventional antibiotics for any of the uses describedherein.

Bacterial Challenge

Three days post-injection, the infected Atlantic salmon were lethargicand anorexic. On sampling, the posterior kidneys of the injected fishwere positive for A. salmonicida whereas those of the control fish werenot.

Molecular Characterisation of Hepcidin cDNAs

Although the winter flounder EST database contains sequences from liver,ovary, stomach, intestine, spleen and pyloric caecae cDNA libraries andthe Atlantic salmon EST database contains sequences from liver, headkidney and spleen, hepcidin-like sequences were only detected in spleenand liver cDNA libraries of both fish. Four of 135 ESTs (3.0%) in thewinter flounder liver library and two of 281 ESTs (0.7%) in the winterflounder spleen library encoded hepcidins. Three of 982 (0.3%) ESTs inthe Atlantic salmon liver library encoded hepcidins. Five hepcidinsequences were also found in subtracted spleen (1.8%) and three insubtracted liver (0.6%) Atlantic salmon cDNA libraries that wereenriched in transcripts up-regulated during infection with Aeromonassalmonicida. Unfortunately, since these are subtracted libraries, theinserts are only portions of the complete transcripts.

Analysis of the nucleotide sequences of Atlantic salmon hepcidin cDNAsrevealed that one salmon EST (SL1-0412) was approximately 300nucleotides longer than the other two. Furthermore, the hepcidin codingsequence was incomplete. Complete sequencing of this clone revealed thepresence of two introns with standard GT/AG splice junctions (FIG. 16A).When removed, an open reading frame encoding a complete hepcidin-likepeptide was obtained. Similarly, an incompletely spliced halibuttranscript was amplified that still retained the second intron (FIG.16B). Compared to mammals, the introns of salmon and probably halibutare in similar locations but of shorter length (FIG. 16C). In additionto these incompletely spliced cDNAs, we identified a winter flounder EST(WF4) that contains a large deletion relative to the other sequencesthat corresponded closely to the second exon of salmon and humanhepcidin. Assuming the intron positions are conserved among vertebrates,this deletion could correspond to the removal of exon 2, and resulted ina peptide that differed from WF3a and WF3b in only five amino acidpositions of the remaining peptide.

FIG. 16 describes a Nucleotide sequence of unspliced liver cDNA encodingType I salmonid hepcidin. Exon sequences are indicated in upper caseletters and the deduced amino acid sequence is shown below thenucleotide sequence. The gt/ag intron/exon boundaries are highlighted inboldface and the polyadenylation signal (aataaa) is underlined. B.Nucleotide sequence of partially spliced cDNA from halibut spleenencoding Type I salmonid hepcidin. C. Comparison of intron/exonstructure in human, mouse and salmon. Exons are represented by hatchedboxes and introns by a single line (sizes in bp shown beneath).

The deduced amino acid sequences of five different winter flounderhepcidin cDNAs and two different Atlantic salmon hepcidins were alignedfor comparison purposes with those extracted from dbEST corresponding toJapanese flounder (two), medaka (one) and rainbow trout (one), as wellas the recently reported hepcidin from hybrid striped bass (Shike et al.2002) and two from Atlantic halibut (Hb 17 and Hb 357). The sequencesobtained from spleen and liver of Atlantic salmon (Sal2.1 and Sal8.6)and Atlantic halibut (Hb1.1, Hb5.3 and Hb7.5) by PCR are also included(FIG. 17). Human hepcidin was included as a representative of themammals. The position of cleavage by signal peptidase was predicted byPSORT and the RX(K/)R motif typical of propeptide convertases (Nakayama1997) was identified (vertical arrows; FIG. 17). The signal peptidesequence is 22-24 amino acids and is highly conserved among all of thefish sequences. The anionic propiece is 38-40 amino acids, depending onthe particular hepcidin variant. The processed hepcidins contain 19-27amino acids and all are positively charged at neutral pH except WF2(Table 10). Types I and III hepcidin from flatfish as well as salmontype hepcidin contain eight cysteine residues in the mature peptide,which have been proposed to form four disulphide bonds. Type II winterflounder hepcidin is missing two cysteine residues, indicating that amaximum of three disulphide bonds could form. Hb357 contains only fivecysteine residues and is quite different from the remaininghepcidin-like sequences. Results of secondary structure predictionmethods indicated that the consensus structure of fish hepcidins wasmostly random coil, although short stretches of extended strand werepredicted by some methods.

FIG. 17 describes Alignment of winter flounder (WF1, WF2, WF3a, WF3b,WF4), Atlantic halibut (Hb1.1, Hb5.3, Hb7.5, Hb17, Hb357) and Atlanticsalmon (Sal1, Sal2, Sal2.1, Sal8.6) hepcidins with those of Japaneseflounder (JFL4, JFL6), medaka, hybrid striped bass and human. A partialsequence from rainbow trout (GenBank accession AF281354_(—1)) is alsoshown. The predicted positions of signal peptidase and pre-proteincleavages are indicated by arrows.

From FIG. 17, it is apparent that all of the flatfish-type hepcidinshave very similar signal peptides, which differ somewhat from thesalmonid type and human hepcidin. Other novel features identifiedincluded different groups of hepcidins based on (1) number of cysteines,(2) unique insertion FKC in flatfish Type III, (3) two other locationsthat may contain unique insertions (4) a truncated version (FlatfishType IV), (5) longer versions at the amino terminus.

Based on the alignment, it is apparent that there are at least threedifferent groups of flatfish hepcidins distinguishable by sharedinsertions and deletions. WF2 and JFL6 (Flatfish Type II) share adeletion of seven amino acids near the KR cleavage site resulting in aprocessed peptide of 19 amino acids, whereas WF3a, WF3b, WF4, Hb1.1,Hb17, Hb5.3 and Sal8.6 (Flatfish Type III) exhibit a deletion of onlyfour amino acids (excluding the portion corresponding to the missingexon of WF4) resulting in processed peptides of 22 amino acids. WF1 andJFL4 (Flatfish Type I) do not contain this deletion but do contain aninsertion relative to all other reported hepcidins at a positionadjacent to the signal peptidase cleavage site. In addition, WF1, bassand medaka share an insertion of one amino acid within the maturepeptide relative to all other reported hepcidins, giving a peptide of26-27 amino acids. WF3a and WF3b differ from each other by only oneamino acid although they contain several silent substitutions anddifferences in the 5′ and 3′ untranslated regions. Hb357 represents apossible fourth class of flatfish hepcidins. The 3′ untranslated regionsof WF2 and WF1 are very different from those of the other hepcidintranscripts, WF2 containing a long additional portion relative to theothers and WF1 being shorter and less highly conserved (FIG. 18A).

The salmonid hepcidin-like peptides fall into one group; the fourreported sequences all share two deletions and differ from each other byfour amino acids in the mature peptide and four amino acids in theupstream pre-protein portion. The 3′ untranslated regions of the salmonhepcidins are only moderately conserved (FIG. 18B).

FIG. 18 describes Alignment of 3′ untranslated regions of (A) winterflounder (WF1, WF2, WF3a, WF3b, WF4) and (B) Atlantic salmon (Sal1,Sal2) hepcidin cDNAs. Conserved nucleotides are boxed. The positions ofthe primers used to amplify hepcidin homologs from halibut and salmonare indicated by arrows.

Genomic Organisation of Winter Flounder Hepcidin Genes

Southern hybridization analysis of genomic DNA from a wide variety offish with a probe corresponding to Type I hepcidin identified bands inall flatfish tested but none of the other fish species (FIG. 19). Inwinter flounder, two fragments of 4.3 and 4.5 kb hybridized with theprobe. Two fragments of yellowtail flounder of identical size hybridized(4.3 kb) and two fragments of witch flounder genomic DNA also hybridized(4.3 and 20 kb), whereas only one fragment (4.3 kb) of the Americanplaice and one fragment (5.5kb) of the Japanese flounder genomic DNAhybridized.

FIG. 19 describes Southern hybridization analysis of hepcidin indifferent fish species. SstI digests of genomic DNA (7.5 μg) fromhagfish (Hg), shark (Sh), white sturgeon (St), winter flounder (WF),yellowtail flounder (YF), American plaice (AP), witch flounder (Wi),Japanese flounder (JF), Atlantic salmon (AS), smelt (Sm) and haddock(Hd) were hybridized with Type I hepcidin from winter flounder. Sizemarkers (M) are Lambda DNA digested with StyI.

Identification of Hepcidin-Like Sequences by RT-PCR

FIG. 2 describes amplification of hepcidin cDNAs from halibut and salmonliver and spleen. RNA was prepared from tissues of fish infected with abacterial pathogen to induce expression of antimicrobial peptide genes,reverse-transcribed and subjected to PCR using the primers listed below.Actin was run as a control to show expression of a house-keeping gene.The labelling on the figure is as follows: HL−halibut liver; SL—salmonliver; HS—halibut spleen; SS—salmon spleen; M—markers. For the primers5′U is the Universal 5′ primer used in all reactions, Sal is Hc Sal(below) and WF is HcPA3b (below). (SEQ ID NO: 324) HepUniversal 5′:AAGATGAAGACATTCAGTGTTGCA (SEQ ID NO: 325) HcPA3 3′B2:GTTGTTGGAGCAGGAATCC (SEQ ID NO: 326) Hc Sal: TGCTGGCAGGTCCTCAGAATTTGC

The results of RT-PCR assays of tissue-specific expression of the threewinter flounder hepcidins are shown in FIG. 20. Type I hepcidin wasabundantly expressed in the liver and, to a lesser extent, in thecardiac stomach. Type II hepcidin could not be detected in any tissues,whereas Type III hepcidin was moderately expressed in the esophagus,cardiac stomach, and liver. In uninfected Atlantic salmon, Type Ihepcidin was expressed at quite high levels in the liver, blood andmuscle, at low levels in gill and skin, and at barely detectable levelsin anterior and posterior kidney (FIG. 21A, Table 10). Type II hepcidinwas expressed at barely detectable levels in the gill and skin only(FIG. 21B). However, fish infected with Aeromonas salmonicida showedexpression of both types of hepcidin in most tissues tested (see below).

RT-PCR analysis of hepcidin gene expression in winter flounder larvae ofdifferent ages is shown in FIG. 22. Transcripts of Type II hepcidinscould not be detected at any stage of development, whereas Type I andType III hepcidins were detectable in pre-metamorphic larvae. Type Ihepcidin was more abundantly expressed than Type II hepcidin and wasalso expressed at an earlier time (5 dph vs. 9 dph.).

FIG. 20 describes Reverse transcription-PCR assay of hepcidin and actingene expression in different tissues of winter flounder. Amplificationproducts from adult winter flounder were amplified using gene-specificprimers for Flatfish Type I (panel A), Type II (panel B) and Type III(panel C) hepcidins and for actin (310 bp) and resolved byelectrophoresis on a 2% agarose gel. Markers (M) are the 100 bp ladder(BRL)

FIG. 21 describes Reverse transcription-PCR assay of hepcidin and actingene expression in different tissues of control Atlantic salmon (C) andthose infected with Aeromonas salmonicida (I). Amplification productsfrom reactions using gene-specific primers for Salmonid Type I (panel A)and Type II (panel B) hepcidins (163 bp) and for actin (400 bp) wereresolved by electrophoresis on a 2% agarose gel. Markers (M) are the 100bp ladder (BRL).

FIG. 22 describes Reverse transcription-PCR assay of hepcidin and actinexpression in developing winter flounder larvae. Samples were larvae at5 dph (lane 1), 12 dph (lane 2), 19 dph (lane 3), 27 dph (lane 4), 41dph (lane 5) and adult (lane 6). Amplification products from reactionsusing gene-specific primers for Flatfish Type I (panel A), Type II(panel B) and Type III (panel C) hepcidins and for actin (400 bp) wereresolved by electrophoresis on a 2% agarose gel using a 100 bp ladder(Pharmacia) as markers (lane M).

Identification of Additional Hepcidin-Like Sequences From Other FishSpecies

Using a primer based on highly conserved sequences in the signal peptideof all reported hepcidins (Hep Universal 5′) in combination with primersbased on highly conserved sequences in the 3′ UTR of salmon (HcSal 3′)and flatfish (HcPA3b 3′), it was possible to amplify hepcidin-likesequences from the liver and spleen of halibut and salmon (FIG. 2). Analignment of the deduced amino acid sequences of hepcidin-like peptidesfrom winter flounder, Atlantic halibut and Atlantic salmon is shown inFIG. 17. Interestingly, flatfish-type hepcidin could be amplified fromsalmon (S8.6) and salmon-type hepcidin could also be amplified from aflatfish (Hb7.5). Additional sequences were obtained from genomic DNA ofPetrale sole, C—O sole, English sole, starry flounder, European plaice,Greenland halibut and Pacific halibut.

FIG. 17 depicts an alignment of certain winter flounder (WF1, WF2, WF3a,WF3b, WF4) Atlantic halibut (Hb1.1, Hb5.3, Hb7.5, Hb17, Hb357) andAtlantic salmon (Sal1, Sal2, Sal2.1, Sal8.6) hepcidins with those ofJapanese flounder (JFL4, JFL6, medaka, hybrid striped bass and human. Apartial sequence from rainbow trout (Genbank Accession AF281354_(—1)) isalso shown. The predicted positions of signal peptidase and pre-proteincleavages are indicated by arrows.

Specific non-limiting examples of hepcidin sequences identified areshown in Table 11. Examples of cDNA or genomic sequences are shown inTable 13.

Pleurocidins

Most antimicrobial peptides, including cecropins and dermaseptins, areencoded by multigene families that have probably arisen by sequentialgene duplications. We have demonstrated that the winter flounder, andprobably other flatfish, possess a gene family encoding antimicrobialcompounds similar to pleurocidin. Comparison of the genomicamplification products obtained using PL1/2 with the cDNA sequence (FIG.1A) showed that WF2 and WF4 contain three introns, the first of whichoccurs only 1 bp upstream from the initiator methionine. The second andthird introns both occur within the mature peptide. The genes for GLa,xenopsin, levitide and caerulein—all skin peptides from Xenopuslaevis—also contain an intron 1 bp upstream from the initiatormethionine (Kuchler et al 1989). The intron positions are conserved inall but WF3 (FIG. 6), but they differ dramatically in size (Table 5),indicating that a considerable period of evolutionary time has elapsedsince the duplication events occurred, or that the intron sequences arerelatively free to drift.

Southern analysis shows that WF1-4 probes hybridise to other flatfishDNAs, including yellowtail flounder, Atlantic halibut and Americanplaice, but not to haddock, smelt or pollock. This hybridisation couldbe due to the highly conserved signal sequence and anionic portion whichwe have shown to be conserved in sequences isolated from these flatfish.Flatfish may provide a rich reservoir of potential therapeutants for theaquaculture industry. The probes for the different pleurocidin familymembers often recognise the same restriction fragments in winterflounder DNA, indicating that they may be clustered at a single locus onthe genome. Complete sequencing of two Lambda clones hybridizing topleurocidin confirms that such clustering does in fact occur (FIG. 11).Clustering of antimicrobial peptide genes has also been noted for insectcecropins (Gudmundson et al. 1991) and apidaecins (Casteels-Jossen etal. 1993), among others.

FIG. 11 describes an embodiment of a Schematic of genomic organizationof pleurocidin-like genes and pseudogenes (ψ) from winter flounder.Introns are represented by solid boxes and exons by stippled boxes.

All of the members of the pleurocidin family are encoded asprepropolypeptides consisting of an amino-terminal signal sequencefollowed by the active peptide and ending with an acidic portion. Thededuced amino acid sequences of the signal and acidic sequences are veryhighly conserved whereas those of the predicted mature antimicrobialpeptides are more variable (FIG. 6). All, however, appear to fold intoamphipathic α-helices. This sequence conservation has allowed us to usea genomic approach to identify many different members of the pleurocidingene family, not only from winter flounder but also from a variety ofother flatfish (FIG. 3, Table 4, Appendix I).

The structure of the pleurocidin prepro polypeptides bears certainresemblances to the frog dermaseptin precursors, which also contain asignal sequence of similar length (22 amino acids) and an acidic portionof 16-25 amino acids. From the full-length cDNA clone (FIG. 1A), theacidic portion of pleurocidin was shown to contain 21 residues. A majordifference between the pleurocidin and dermaseptin prepolypeptides isthe position of the acidic portion—downstream of the mature peptide inpleurocidin and upstream of the mature peptide in dermaseptins. Theacidic proparts of defensins have been proposed to prevent interactionof the antimicrobial peptide with the membrane by neutralising thecationic charges (Valore et al. 1996) and this may also be its functionin pleurocidin. This feature can be of practical significance fordelivering peptides that are inactive until specifically cleaved.

The signal sequences and acidic carboxy-terminal sequences of thepleurocidin family members are extremely highly conserved. The former,and possibly the latter, are presumed to target the precursor moleculesto the cell membrane for secretion. Gene families for antimicrobialpeptides that contain highly conserved signal peptides (often encoded bythe first exon) followed by end products with different biologicalactivities have been described from the dermaseptin family (Valore etal. 1996) and the GLa, xenopsin, levitide and caerulein, all of whichare skin peptides from Xenopus laevis (Kuchler et al. 1989). Theseauthors proposed that this modular gene structure allows targeting forsecretion to be achieved for markedly different peptides using a commonpathway. In the pleurocidin gene family, a modular structure is alsopresent with exon 2 encoding the signal sequence and first half of theantimicrobial peptide, exon 3 encoding the next ten amino acids of theantimicrobial peptide, and exon 4 encoding the last three amino acids ofthe antimicrobial peptide and the acidic carboxy terminus.

The mature peptides encoded by WF2 and WF4 are 60% identical to eachother (FIG. 6) and somewhat less similar to dermaseptin B I andceratotoxin B (Cole et al. 1997). WF1 is 64% identical to WFla butcontains a remarkably cationic stretch of 18 amino acids between thesignal sequence and the mature peptide that is not present in WF1a.Whether or not this potentially antimicrobial 18-mer peptide arises whenpleurocidin WF1 processing occurs remains to be determined. Both WF1 andWF1a contain an additional 10-11 amino acids relative WF2, WF3 and WF4between the mature peptide and the acidic carboxy terminus. WF3 sharessimilarities with both WF2/4 and WF1/1a. Synthetic pleurocidin identicalto the central portion of WF2 has been shown to protect Coho salmonagainst infection by Vibrio anguillarum, as have hybrid peptides basedon pleurocidin, dermaseptin and ceratotoxin (Jia et al. 2000).

The tissue-specific expression of the pleurocidin genes was assessedusing northern blot analysis and RT-PCR. Northern analysis proved to benot sufficiently sensitive for detecting the low level of transcriptspresent in winter flounder mRNA. Transcripts were present only in skinin sufficient quantities to be detected by this method, so the moresensitive RT-PCR assay was used. Pleurocidin transcripts were found inboth skin and intestine using this method, in agreement with therecently reported ultrastructural localisation of pleurocidin in thesetissues (Cole, Darouiche et al. 2000) and supporting the role ofpleurocidin in mucosal immunity. The transcript size (approximately 200bp) is consistent with the size of products obtained by RT-PCR (Table7), showing that the pleurocidin genes are transcribed separately.

RT-PCR analysis showed that the genes for the different pleurocidin-likepeptides are expressed in a tissue-specific manner with WF2 beingexpressed predominantly in the skin and gill and to a lesser extent inthe muscle, intestine, stomach and liver whereas WF1 and WF4 aredetected predominantly in the gill and skin (FIG. 7). WF3 and WFYT areexpressed in most of the tissues sampled, WFX is detected solely in theskin and WF I a was not expressed in any of the tissues sampled.Possibly, the different antimicrobial peptides are required to controlthe growth of different bacterial populations in the two tissues. Sinceno RT-PCR products were detected for WF4, it is possible that this geneis expressed only at low levels in adult skin or intestine or that it isexpressed at a different life stage or in a different tissue.

Using primers that did not discriminate between the transcripts of thevarious pleurocidin-like genes, expression was first detected at 5 dphand showed a progressive increase towards adulthood. However, recentexperiments using primers specific for WF1, WF1a, WF2, WF3, WF4, WFX andWFYT, transcripts were detected at different developmental stages (FIG.9). WFX was only detectable at 20 dph, whereas WFYT, WF3 and WF2 weredetectable at 5 dph and at higher levels between 25-36 dph.Interestingly, WF1 was not detectable at any larval stage and may onlybe expressed under specific environmental conditions in response tospecific bacterial pathogens, as has been shown for Drosophila (Rivasand Ganz 1999). This is the first demonstration of developmentalexpression of an antimicrobial peptide in fish and shows that at leastthis component of innate immunity is present in early larval stages ofwinter flounder. Larval mortality prior to metamorphosis is of greatconcern and although the reasons for such mortality are not yet known,high bacterial load in the gut has been proposed (Padros, Minkoff et al.1993). The adaptive immune systems of flatfish have been shown todevelop later than those of other teleosts (Padros, Sala et al. 1991).Thus, the ability of larvae to produce antimicrobial peptides duringthis period may be crucial to survival, and the identification offactors that increase the production of such compounds would be of greatbenefit to aquaculturalists.

These results of testing synthetic peptides against a variety ofbacterial pathogens as well as the fungal pathogen, Candida albicans,show promising candidates with broad-spectrum antimicrobial activities.Of particular interest is the ability of the peptides NRC-13 and NRC-15to inhibit the growth of methicillin-resistant S. aureus atconcentrations as low as 4 μg/ml. NRC-13 is also capable of inhibitingthe growth of C. albicans at 4 μg/ml, P. aeruginosa at 1 μg/ml (andkilling P. aeruginosa at this concentration), and A. salmonicida at 2μg/ml. This means that NRC-13 is highly active against a fish pathogen,a Gram-negative human bacterium, a drug-resistant Gram-positive humanbacterium, and a yeast. The example of NRC-13 demonstrates the range ofpotential targets and applications for cationic antimicrobial peptides.

These results also validate the process we used for selectingantimicrobially active peptides from a large amount of sequence data.The ability to accurately predict which peptides are likely to be activeis a crucial link between genomics and therapeutics. While much workremains to be done in this area, we have clearly demonstrated thatjudicious application of the principles described earlier will aid inselecting active peptides.

Thus, a variety of cDNA and genomic sequences encoding the precursors ofantimicrobial peptides identical to or similar to pleurocidin from avariety of flatfish species have been isolated. Northern hybridisationand sequence analysis of RT-PCR products showed that expression wastissue-specific. Most importantly, the timing of expression of differentpleurocidin variants in developing larval winter flounder wasdetermined, allowing an estimate of the onset of the innate immunesystem in this fish. These assays of pleurocidin expression are usefulin directing the screening strategy for isolating novel peptidesequences expressed during specific tissues and/or developmental stages.Environmental parameters affecting the production of pleurocidin canalso be assayed.

This work paves the way to further studies aimed at the over-expressionof pleurocidin as a therapeutant for aquacultured fish and theproduction of disease-resistant fish through transgenic technology ashas been demonstrated in transgenic tobacco expressing antimicrobialpeptides (Jach et al. 1995) and proposed for fish (Jia et al. 2000).Furthermore, because many fish live in a saline environment, theproperties of their antimicrobial peptides may be different from thoseproduced by terrestrial animals and have application in uniquesituations. For instance, the pulmonary mucosa of patients with cysticfibrosis contain elevated NaCl concentrations, which inhibit the naturalcationic peptides secreted by the lung (Goldman et al. 1997).Salt-adapted cationic peptides from marine fish may have application inthe treatment of lung infections in these patients.

Hepcidins

Sequence analysis of one salmon EST (SL1-0412) and one halibut clone(Hb7.5), revealed the presence of unspliced transcripts and allowed thepositions of some of the introns to be determined (FIG. 16). Similar tomouse, human and hybrid striped bass, the salmon hepcidin is composed ofthree exons and two introns (Park, Valore et al. 2001; Shike et al.2002; Pigeon, Ilyin et al. 2001). The position of the first intron ofsalmon and bass are identical and correspond to a position two aminoacids 5′ to those of mouse and human. However, the second salmon intronand the second halibut intron of Hb7.5 correspond to a position twoamino acids 3′ to those of mouse and human and several amino acids 5′ tothat of the bass. This is probably due to “intron sliding” whereby thepositions of introns have shifted by several nucleotides over the courseof evolution. Interestingly, the deletion in WF4 corresponds preciselyto the position of the first salmon intron and the second mouse/humanintron, indicating an intermediate intron/exon structure.

Mouse contains two hepcidin genes that are clustered on the genome(Pigeon, Ilyin et al. 2001) but in human (Park, Valore et al. 2001) andstriped bass (Shike et al. 2002) only one hepcidin gene has beenidentified. Although the number of hepcidin genes in winter flounder andAtlantic salmon remains to be determined, there are at least five inwinter flounder, five in Atlantic halibut and four in Atlantic salmon.Since there are no SstI sites within the hepcidin probe used in theSouthern hybridization analysis, it is highly probable that the fivewinter flounder hepcidin genes reported here are clustered on twogenomic fragments. Multiple genes for pleurocidin also exist (Douglas,Gallant et al. 2001) and are clustered on the genome (FIG. 11).Interestingly, all of the small flounders tested from the Atlanticexhibited a similar hybridizing band of 4.3 kb, indicating that theyshare similarity at the genomic level. Japanese flounder, found in thePacific, exhibited a single hybridizing band of 5.5 kb.

The deduced amino acid sequences of the fish prepro-hepcidins can bealigned with those from mammals throughout their length but only showhigh similarity in the portion corresponding to the processed peptides(FIG. 17). However, within the fish, the signal peptide and the propieceare also very highly conserved. Conservation of these segments has alsobeen noted in the pleurocidin family (Douglas, Gallant et al. 2001). Theamino-termini of the processed peptides were assigned based on the aminoacid sequence of human hepcidin (Krause, Neitz et al. 2000; Park, Valoreet al. 2001) and the proximity to the RX(K/R)R motif characteristic ofprocessing sites (Nakayama 1997). The molecular weights of the processedhepcidins from winter flounder and Atlantic salmon range from 1992 Da(WF2) to 3066 (WF1), comparable to hepcidins isolated from mouse, humanand bass. With the exception of WF2, which has an acidic pI (5.54), thepIs of hepcidins are between 7.73 and 8.76.

Like pleurocidins, the amino acid sequences of the hepcidin variants arehighly similar within species, suggesting relatively recent duplicationof an ancestral gene. It is possible that the aquatic environment inwhich fish live necessitates the existence of a more diverse suite ofantimicrobial peptides than in terrestrial mammals. In addition, thiscomponent of the innate immune system plays a more major role in fishthan in mammals, which have a more highly evolved adaptive immunesystem.

The human hepcidin molecule has been proposed to form a secondarystructure containing a series of β-turns, loops and distorted β-sheets(Park, Valore et al. 2001). Consensus secondary structure prediction offish hepcidins show that they contain mostly random coil structure withsome extended strand structure. With the exception of WF2, JFL6 andHb357, all hepcidins reported thus far contain eight cysteine residueswhich are proposed to form four disulphide bonds (Krause, Neitz et al.2000; Park, Valore et al. 2001) in the following linkage pattern: 1-4,2-8, 3-7, 5-6 (Park, Valore et al. 2001). The loss of cysteine residues1 and 3 from WF2 suggests that at least one disulphide bond cannot form.

Using gene-specific primers, we were able to demonstrate that differenthepcidin genes are expressed in different tissues of both winterflounder (FIG. 20) and Atlantic salmon (FIG. 21). In Atlantic salmon,hepcidin was detectable in normal uninfected fish predominantly inliver, blood and muscle (Type I) and to a lesser extent in gill and skin(both types). This is consistent with the presence of three ESTs forType I hepcidin in cDNA libraries constructed from uninfected livers,and the absence of ESTs for Type II hepcidin in cDNA librariesconstructed from uninfected liver, spleen and head kidney. Type IIhepcidin expression appears be confined to external epithelial surfacesin contact with the aqueous environment, whereas Type I hepcidinexpression is more widespread, being expressed in liver, blood andmuscle as well as external epithelial surfaces. In uninfected winterflounder, no transcripts of Type II hepcidin could be detected in anytissue but transcripts of Types I and III hepcidin were present in theliver and cardiac stomach. Type III hepcidin transcripts were alsopresent in the esophagus.

Mouse hepcidin was also reported to be predominantly expressed in liver,and weakly in stomach, intestine, colon, lungs, heart and thymus byNorthern analysis using one of the mouse hepcidin sequences as probe(Pigeon, Ilyin et al. 2001). However, this study did not discriminatebetween the two hepcidin genes and it is not known whether or not thetwo mouse genes are differentially expressed in tissues of mouse.Similarly, dot-blot analysis of human tissues and cell lines using thehuman hepcidin cDNA as probe revealed strong expression in adult andfetal liver and weaker expression in adult heart, fetal heart and adultspinal cord (Pigeon, Ilyin et al. 2001). An earlier study using RealTimequantitative RT-PCR (Krause, Neitz et al. 2000) revealed strongexpression of hepcidin in human liver, heart and brain and weakexpression in a variety of other tissues. Interestingly, we could notdetect either Type I or Type II hepcidin expression in the brain ofnormal Atlantic salmon or winter flounder, or heart of normal Atlanticsalmon. However, in infected animals, Type II hepcidin was expressed inboth tissues, indicating that this form is the predominant one producedunder conditions of stress.

It is intriguing that we detected transcripts of Type I hepcidin thatwere constitutively expressed in blood cells of Atlantic salmon.Constitutively expressed non-enzymic antimicrobial molecules have beenreported only rarely in blood of fish; a small hydrophobic cationicpeptide was found in mucus of rainbow trout (Smith et al., 2000) andmoronecidin, an antimicrobial peptide from bass, was expressed in bloodof uninfected animals (Lauth et al. 2002). Interestingly, expression ofneither hepcidin increased in blood of infected salmon relative to theuninfected control animals. Possibly, hepcidin is fulfilling a role iniron homeostasis in control animals as well as an antimicrobial role.Its presence in circulating blood cells of uninfected animals may be aprecautionary measure against impending infection.

Type I and II hepcidins from Atlantic salmon were up-regulated duringinfection with Aeromonas salmonicida, but to different extents invarious tissues. While Type I hepcidin was noticeably up-regulated inthe esophagus, stomach, pyloric caecae, liver, spleen, intestine,posterior kidney, rectum and muscle and to a lesser extent in anteriorkidney and skin, Type II hepcidin showed a more dramatic increase instomach, pyloric caecae, liver, spleen, intestine, brain, heart andmuscle. Weaker up-regulation was present in esophagus, anterior andposterior kidney, skin and rectum. These results are consistent withthose reported for bacterially challenged hybrid striped bass whereup-regulation was most dramatic in liver, but was also demonstrated inskin, gill, intestine, spleen, anterior kidney and blood (Shike et al.2002). It is not known whether there are multiple hepcidins in hybridstriped bass and, if so, whether they are differentially expressed as inAtlantic salmon and winter flounder.

Studies with mice have shown a 4.3-fold increase in hepcidin expressionin livers of mice injected with LPS and a 7-fold increase in primaryhepatocytes exposed to LPS (Pigeon, Ilyin et al. 2001). These studieswere based on Northern analysis using only one of the mouse hepcidinsequences as probe, and were therefore unable to distinguish possibledifferential expression of the two mouse variants. Similar increaseswere noted in livers of mice subjected to iron overload, but not forprimary hepatocytes exposed to iron citrate, possibly due to thedifferentiation status of the cultured hepatocytes. The fact that bothiron overload and LPS exposure increase hepcidin expression indicatesthe importance of these two factors in the host response to pathogens.

During infection, iron is removed from the system by various mechanismsso that it is unavailable for use by invading pathogens. It has beenproposed that recently discovered transferrin receptor2 mediates ironuptake by hepatocytes and increases their expression of hepcidin(Fleming and Sly 2001; Nicolas, Bennoun et al. 2001). Hepcidin, in turn,increases iron accumulation in macrophages and increases dietary ironabsorption in duodenal crypt cells via β2 microglobulin, HFE andtransferrin receptor1. These crypt cells differentiate into enterocyteswith reduced amounts of iron transport proteins, thereby decreasingdietary iron uptake. Hepcidin thus appears to play a crucial role iniron homeostasis during inflammation as well as acting as anantimicrobial peptide. It is also possible that hepcidin could modulateexpression of liver-derived acute phase proteins and exhibit synergisticeffects with other components of the immune system.

Antimicrobial peptides have been shown to modulate gene expression inmouse macrophages (Scott, Rosenberger et al. 2000), and it is possiblethat they may exert similar effects in fish macrophages or hepatocytes.The presence of a functional nuclear localization signal (four K/Rresidues in a row) within prohepcidin of mouse and human indicates thathepcidin could act as a signaling molecule involved in maintenance ofiron homeostasis in these organisms (Pigeon, Hlyin et al. 2001).Interestingly, the nuclear localization signal also contains therecognition signal for processing of prohepcidin, indicating thatnuclear localization would occur only prior to removal of the propiece,or that the propiece itself is localized to the nucleus. Teleosthepcidins contain only 3 out of 4 K/R residues, which may not besufficient for nuclear localization; a role for hepcidin inintracellular signaling awaits testing with synthetic or invitro-expressed peptide.

In conclusion, the sequences of new hepcidin-like peptides fromdifferent fish species and the presence of related sequences in severalflatfish species by Southern hybridization have been determined.Furthermore, it has been shown that the various types of fish hepcidinsare differentially expressed in a tissue-specific manner in normal fish,as a result of bacterial infection, and during larval development, thusproviding a strategy for identifying additional sequences for novelpeptides. Apparently in fish, different tissues produce hepcidins in aconstitutive or inducible manner, indicating that hepcidin variants mayhave different functions under different circumstances. Given their rolein iron homeostasis in mammals, it is possible that fish hepcidinvariants may fulfill this role as well as that of killing specificpathogens. In vitro expression of hepcidin variants will allow theirspectrum of antimicrobial activity to be determined as well as theireffect on the innate immune response.

Thus, there has been provided a method for identifying potentialantimicrobial peptides.

Tables

Table 1. Nucleotide sequences of oligonucleotides used for isolatingpleurocidin-like sequences (SEQ ID NOS: 1-10, left to right, in order ofappearance)

Table 2. Nucleotide sequences of oligonucleotides used for assay ofpleurocidin-like gene expression in different tissues and at differentstages of development of winter flounder (SEQ ID NOS: 11-34, left toright, in order of appearance)

Table 3. Nucleotide sequences of primers used in RT-PCR assays toanalyse hepcidin gene expression. (SEQ ID NOS: 35-61, left to right, inorder of appearance) The amino acid sequence on which the 5′ primer wasbased is shown. The 3′ primers were within the 3′ untranslated region(3′ UTR). The annealing temperatures used in the PCR reactions and thesizes of the amplification products are listed.

Table 4. One-letter amino acid sequences for pleurocidins based ongenomic and expression data (SEQ ID NOS: 62-81, respectively, in orderof appearance)

Table 4a. Bacterial and Candida strains used in this study

Table 5. Sizes of introns (in bp) in genomic sequences amplified usingprimers PL5′ and PL3′

Table 6. RT-PCR products from skin and intestine corresponding todifferent pleurocidin genes

Table 7. Sizes of bands (in kb) hybridising to pleurocidin probes inBamHI and SstI digests of winter flounder DNA

Table 8. Minimal inhibitory concentrations of pleurocidin-like cationicantimicrobial peptides against a wide spectrum of bacterial pathogensand Candida albicans.

Table 9. Characteristics of winter flounder and Atlantic salmonhepcidin-like peptides

Table 10. Results of PCR analysis of hepcidin expression

Table 11. One-letter amino acid sequences for certain hepcidins based ongenomic and expression data, including NRC reference numbers (SEQ IDNOS: 174-211, respectively, in order of appearance)

Table 12. Nucleotide sequences of pleurocidin-like peptides of Table 4

Table 13. Nucleotide sequences of hepcidin-like peptides of Table 11.

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Wu, M., E. Maier, R. Benz, and R. E. W. Hancock. 1999. Mechanism ofinteraction of different classes of cationic antimicrobial peptides withplanar bilayers and with the cytoplasmic membrane of Escherichia coli.Biochem. 38:7235-7242. TABLE 1 Nucleotide sequences of oligonucleotidesused for isolating pleurocidin-like sequences Amino Acid Primer SequenceNucleotide Sequence (5′ => 3′) Screening cDNA library PleuroAFFKKAAHVGKH TTCTTCAAGAAGGCYGCYCAYGT[C/G]GG [C/A]AAGCA PleuroBHVGKAALTHYL¹ CAYGT[C/G]GG[C/A]AAGGCYGCYCT[C/G] AA[C/T/A]CAYTACCT GenomicPCR and RT-PCR PL1 5′ untranslated GCCCACTTTGTATTCGCAAG PL2 3′untranslated CTGAAGGCTCCTTCAAGGCG PL5′ MKFTATF ATGAAGTTCACTGCCACCTTCPL3′ KRAVDE¹ TCATCGACTGCGCGCTT¹complement

TABLE 2 Nucleotide sequences of oligonucleotides used for assay ofpleurocidin-like gene expression in different tissues and at differentstages of development of winter flounder Amino Acid Gene Primer SequenceNucleotide Sequence (5′ => 3′) WF1 RTWF1 KGRWLER AAGGGCAGGTGGTTGGAAAGGRTWF1/3′ YQEGEE¹ CCCTCCCCCTCCTGGTA WF1a RTWF1a RKRKWLRCGTAAGAGAAAGTGGTTGAGA RTWF1a/3′ YQEGEE¹ CCCTCCCCCTCCTGGTA WF2 RTWF2KAAHVG AAGGCTGCTCACGTTGGC PL2 3′ untranslated CTGAAGGCTCCTTCAAGGCG WF3RTWF3 FLGALIK TTCTTAGGAGCCCTTATCAAA RTWF3/3′ YDEQQE¹ CTCCTGCTGCTCGTCATAWF4 RTWF4 HGRHAA CATGGTCGTCATGCTGCC PL2 3′ untranslatedCTGAAGGCTCCTTCAAGGCG WFYT RTWFYT GFLFHG GGGATTTCTTTTTCATGG RTWFYT/3′SFDDNP¹ GGGTTGTCATCGAATGAG WFX RTWFX RSTEDI CGTTCTACAGAGGACATC RTWFX/3′DDDDSP¹ GGGGCTGTCATCATCATC

TABLE 3 Nucleotide sequences of primers used in RT-PCR assays to analysehepcidin gene expression. The amino acid sequence on which the 5′ primerwas based is shown. The 3′ primers were within the 3′ untranslatedregion (3′ UTR). The annealing temperatures used in the POR reactionsand the sizes of the amplification products are listed. Type (size)Primer Amino acid Nucleotide sequence Annealing (bp) Product aequence(5′ => 3′) temperature size Winter flounder Type I HcPA1 5′ WMENPTTGGATGGAGAATCCCACC 50° C. 137 HcPA1b 3′ 3′ UTR GTGAGGTTGTGTTGCGGG TypeII HcPA2 5′ GMMPNN GGGATGATGCCAAACAAC 50° C. 180 HcPA2b 3′ 3′ UTRACTTGGACTATGGGCTGAG Type III HcPA3 5′ WMMPNN TGGATGATGCCATACAAC 50° C.118 HcPA3b 3′ 3′ UTR GTTGTTGGAGCAGGAATCC Actin ActF (WF) AALVVDTCGCTGCCCTCGTTGTTGAC 50° C. 312 ActR (WF)* VLLTEAP* GGAGCCTCGGTCAGCAGGAActin F1 VFPSIV GTGTTCCATCCATCGTC 50° C. 194 Actin R1 HTFYNELGAGCTCGTTGTAGAAGGTGT Atlantic salmon Type I HCSS 5′ MHLPEPATGCATCTGCCGGAGCCT 55° C. 163 Hep Liv R 3′ UTR CATTGCAAACATGTACAAACTAGType II Hep Sp F MNLPMH ATGAATCTGCCGATGCA 52° C. 163 Hep Sp R 3′ UTRGGGCAAATTAAAGGCG Actin Act400F IVGRPRHQ TCGTCGGTCGTCCCAGGCATCAG 52° C.400 Act400R GYALPHAI ATGGCGTGGGGCAGAGCGTAACC*complement

TABLE 4 Sequences of pleurocidin-like peptides used for activitytesting. Final peptide sequences and patterns of C-terminal amidationwere selected based on the analysis of translated nucleotide sequencesand on principles described in the text. Origin Amino acid sequence CodeWinter Flounder (1) GKGRWLERIGKAGGIIIGGALDHL-NH₂ NRC-01^(a) WinterFlounder (1a) WLRRIGKGVKIIGGAALDHL-NH₂ NRC-02^(a,d) Winter Flounder(1a-l) GRRKRKWLRRIGKGVKIIGGAALDHL-NH₂ NRC-03^(a,d) Winter Flounder (2)2.1 GWGSFFKKAAHVGKHVGKAALTHYL-NH₂ NRC-04^(a) Winter Flounder (3)FLGALIKGAIHGGRFIHGMIQNHH-NH₂ NRC-05^(a) Winter Flounder (4) 1.1GWGSIFKHGRHAAKHIGHAAVNHYL-NH₂ NRC-06^(a) Yellowtail Flounder YT2RWGKWFKKATHVGKHVGKAALTAYL-NH₂ NRC-07^(b) Winter Flounder XRSTEDIIKSISGGGFLNAMNA-NH₂ NRC-08^(b,c) Winter Flounder YFFRLLFHGVHHGGGYLNAA-NH₂ NRC-09^(b.c) Winter Flounder ZFFRLLFHGVHHVGKIKPRA-NH₂ NRC-10^(b,c) American Plaice AP1GWKSVFRKAKKVGKTVGGLALDHYL-NH₂ NRC-11^(b) American Plaice AP2GWKKWFNRAKKVGKTVGGLAVDHYL-NH₂ NRC-12^(b) American Plaice AP3GWRTLLKKAEVKTVGKLALKHYL-NH₂ NRC-13^(b) Witch Flounder GcSc4C5AGWGSIFKHIFKAGKFIHGAIQAHND-NH₂ NRC-14^(b) Witch Flounder GcSc4B7GFWGKLFKLGLHGIGLLHLHL-NH₂ NRC-15^(b) Witch Flounder GC3.8-tGWKKWLRKGAKHLGQAAIK-NH₂ NRC-16^(b) Witch Flounder GC3.8GWKKWLRKGAKHLGQAAIKGLAS NRC-17^(b) Witch Flounder GC3.2GWKKWFTKGERLSQRHFA NRC-18^(b) Halibut Hb26 FLGLLFHGVHHVGKWIHGLIHGHH-NH₂NRC-19^(b) Halibut Hb18 GFLGILFHGVHHGRKKALHMNSERRS NRC-20^(b)^(a)Peptide predicted from expressed tag and/or expression confirmed byRT-PCR and/or by in situ hybridization.^(b)Peptide predicted from genomic sequence^(c)Pseudogenes^(d)NRC-2 and NRC-3 are both derived from the same sequences with thelatter including an additional N-terminal fragment.

TABLE 4a Bacterial and Candida strains used in this study. Species CodeID Comments Escherichia coli C498, UB1005 Parent of DC2 Escherichia coliC500, DC2 Outer membrane-permeable mutant Escherichia coli C786,CGSC4908 Triple auxotroph (thy, uri, L-his) Salmonella enterica s.Typhimurium C587, 14028S Parent of C610 Salmonella enterica s.Typhimurium C610, MS4252S Supersusceptible strain Pseudomonas aeruginosaH187, K799 Parent of H188 Pseudomonas aeruginosa H188, Z61Supersusceptible strain Enterococcus faecalis C625, ATCC29212 Standardstrain (ATCC) Staphylococcus aureus C622, ATCC25923 Standard strain(ATCC) Staphylococcus aureus C623, SAP017 MRSA clinical isolate (fromTony Chow - VGH) Staphylococcus epidermidis C960, ATCC14990 Standardstrain (ATCC) Staphylococcus epidermidis C621 Clinical isolate (fromDavid Speert - Children's) Bacillus subtilis C971, ATCC6633 Standardstrain (ATCC) Aeromonsa salmonicida 99-1, A449 Field isolate beingsequenced at IMB Aeromonas salmonicida 97-4 Field isolate Candidaalbicans C627, CALB105 Yeast test strain

TABLE 5 Sizes of introns (in bp) in genomic sequences amplified usingprimers PL5′ and PL3′ Gene Exon 1 Intron 1 Exon 2 Intron 2 Exon3 TotalWF1 154 539 31 95 82 901 WF1a¹ 103 ? 31 ? 82 ? WF2² 100 525 31 108 49813 WF3 100 374 19 97 64 654 WF4² 100 230 31 101 49 511¹Intron sizes could not be determined as this sequence is onlyrepresented by an RT-PCR product²Sequences were also amplified using primer PL1 and PL2

TABLE 6 RT-PCR products from skin and intestine corresponding todifferent pleurocidin genes Skin Intestine Size Band 4 n/d¹ 265 bp WF1 52 175 bp WF2 4 9 175 bp WF3 n/d¹ n/d¹ — WF4 n/d¹ 7 215 bp n/d²¹not detected²not detected by genomic PCR (corresponds to WF1a)

TABLE 7 Sizes of bands (in kb) hybridising to pleurocidin probes inBamHI and SstI digests of winter flounder DNA Probe BamHI SstI WF1 >24,6 19, 17, 4.5, 4.4, 3.0, 2.9, 2.2, 1.3, x WF2    6 19, 17, 4.5, 4.4,2.9, x 1.3, x WF3 >24 19, 17, 4.5, x 2.9, x 2.2, 1.3, x WF4 17, 6 19,17, 4.5, 4.4, 2.9, x 2.2, 1.3, 1.2x = no hybridising band evident

TABLE 8 Minimal inhibitory concentrations of pleurocidin-like cationicantimicrobial peptides against a wide spectrum of bacterial pathogen andCandida albicans. Pathogers were grown in Mueller-Hinton broth andexposed to a range of concentrations of the specified peptide. Thelowest peptide concentration which inhibited bacterial growth by atleast 50% was recorded as the minimal inhibitory concentration. A. salA. sal S. typh S. typh P. aeru P. aeru E. coli E. coli E. coli S. epiMRSA C. alb 99-1 97-4 MS4252s 14028s K799 Z61 C786 UB1005 DC2 C621 C623C627 NRC-1 64 64 16 >64 >64 32 32 32 32 >64 >64 64 NRC-2 >128 128 64 >6464 32 64 64 64 >64 >64 >64 NRC-3 2 4 2 8 2 1 2 8 2 8 8 4 NRC-4 2 2 2 168 4 2 4 2 8 8 8 NRC-5 >64 >64 64 >64 >64 32 64 64 >64 32 32 >64 NRC-6 44 4 64 16 4 4 4 2 >64 32 32 NRC-7 N/A N/A N/A N/A N/A N/A N/A N/A N/AN/A N/A N/A NRC-8 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64NRC-9 >64 >64 64 >64 >64 64 64 >64 >64 >64 >64 >64 NRC-10 >64 32 16 >6432 8 32 32 32 32 64 >64 NRC-11 8 8 4 32 32 4 4 16 4 64 >64 32 NRC-12 2 22 8 4 1 2 8 2 8 16 4 NRC-13 4 2 2 8 4 1 2 4 2 4 4 4 NRC-14 32 16 16 >6432 8 16 16 16 16 16 >64 NRC-15 8 16 4 16 8 4 8 8 8 4 4 16 NRC-16 2 1 0.516 4 1 1 2 0.5 16 32 8 NRC-17 2 1 1 8 4 2 1 4 1 32 16 8 NRC-18 >64 12832 >64 >64 64 64 64 64 >64 >64 >64 NRC-19 64 >64 16 64 32 8 32 16 32 8 864 NRC-20 >64 >64 >64 >64 >64 64 >64 >64 >64 >64 >64 >64

TABLE 9 Characteristics of winter flounder and Atlantic salmonhepcidin-like peptides Total Total Molecular Name Amino Acids CysteinesWeight pI WF1 27 8 3066 8.75 WF2 19 6 1992 5.54 WF3 22 8 2367 8.74 WF422 8 2256 8.52 Hb5.3 22 8 2363 8.75 Sal8.6 22 8 2331 8.76 Hb17 22 8 23918.76 Hb1.1 22 8 2391 8.76 Hb357 22 5 2397 7.84 Hb7.5 25 8 2881 8.53Sal2.1 25 7 2925 8.60 Sal1 25 8 2720 7.73 Sal2 25 8 2881 8.53

TABLE 10 Semi-quantitative RT-PCR analysis of hepcidin expression inAtlantic salmon during bacterial challenge Type I Hepcidin Type IIHepcidin Tissue Control Infected Ratio Control Infected Ratio Esophagusnd 0.08 ↑ nd 0.09 ↑ Stomach nd 0.09 ↑ nd 0.27 ↑↑ Pyloric caecae nd 0.14↑ nd 0.37 ↑↑ Liver 1.19 2.36 2 nd 1.45 ↑↑↑ Spleen nd 0.18 ↑ nd 0.41 ↑↑Intestine nd 0.21 ↑ nd 0.33 ↑↑ Brain nd nd 0 nd 0.50 ↑↑ Blood 0.82 0.841 nd nd ˜ Anterior kidney 0.06 0.07 1.2 nd 0.08 ↑ Posterior kidney 0.070.14 2 nd 0.11 ↑ Gill 0.13 0.12 1 0.08 0.07 1 Skin 0.14 0.18 1.3 0.070.09 1.3 Ovary nd nd 0 nd nd 0 Rectum 0.07 0.13 2 nd 0.08 ↑ Heart nd nd0 nd 0.43 ↑↑ Muscle 0.38 0.8 2.1 nd 0.60 ↑↑Pixel densities obtained by densitometry are expressed relative to theactin signal. The ratio of infected: control was calculated wherenumerical values were obtained for both conditions.nd, not detected;↑ weakly up-regulated;↑↑ strongly up-regulated.

TABLE 12 Nucleotide sequences encoding pleurocidin-like peptides ofTable 4. NRC-01 Winter Flounder WF1 (SEQ ID NO: 82)ATGAAGTTCACTGCCACCTTCCTCCTGTTGTTCATCTTCGTCCTCATGGTTGATCTCGGAGAGGGTCGTCGTAAGAAAAAGGGGTCGAAGAGAAAGGGGTCCAAGGGAAAGGGGTCCAAGGGAAAGGGCAGGTGGTTGGAAAGGATTGGTAAAGGTAGAGTCACGGAATTAATTTGCTTTTTACATTGCAAATATTTTTCATATAACATTGCTGGAAAATCACAAAAATAAGTAGTCAATATATTTGGCCAAATAGAATCACTTTGATTTCAATAATAATCAAAATAACAACCTAAAAGGCCTTTGATTAGCATGTTCCTTCAATGAAATGGACATTGTAATTTACTTTGATTCTCACATGCTACGACCTGCTGCAGCAACATTTGAAAATAAATTTGTCCCAGAAGATTTTAAAGTACATTGTTATAGGCGATTTATCTTTCTATTACTCAGATATTTGTTCAAACCAATAGAATAACTGGATCTCTATGCTAAAATAATAAAACACACATTCAGATGTTACCAGTCAAGATTGAACGCTGTTTAAAAGTAAGTATGAAACATCCTCTGTATGTATAATTGTTTAACTGGTAACTTATAGTCCTAATAATTGCGTTATGGAAATGTATTAATTGTCATTTAATATAATTTGCTGGAATTTATCACTGTGTGTTTTTGTTTGTTTTTACACAGCTGGCGGGATAATTATCGGGGGGGCCCTTGAGTAAGGACTTCTACCATCATTACTGTGTAATATTTATAGTTATGATCAGTACAGTTATTAACAACTTCTCTTGTCTCGCTGAACTTCTCCATCAGTCACCTCGGGCAGGGGCAGGTGCAGGGGCCGGATTACGACTACCAGGAGGGGGAGGAGCTCAACAAGCGCGCAGTCGATGAA //NRC-02 and NRC-03 Winter Flounder WF1A (SEQ ID NO: 83)ATGAAGTTCACTGCCACCTTCCTCCTGTTGTTCATCTTCGTCCTCATGGTTGATCTCGGAGAGGGTCGTCGTAAGAGAAAGTGGTTGAGAAGGATTGGTAAAGGTGTCAAGATAATTGGCGGGGCGGCCCTTGATCACCTCGGGCAGGGGCAGGTGCAGGGGCAGGATTACGACTACCAGGAGGGGCAGGAGCTCAACAAGCGCGCAGTCGATGAAA //NRC-04 Winter Flounder WF2 (SEQ ID NO: 84)GCCCACTTTGTATTCGCAAGGTAATATTGATATTTTTCATATTCATTTAGACAAATGTGCTCAGCTTGTTACTGTATAATGCAAAAGTTAATGATCTTTATTTTTCTGTTTTTTTTTGTAGAATGAAGTTCACTGCCACCTTCCTCATGATTGCCATCTTCGTCCTCATGGTTGAACCTGGAGAGTGTGGCTGGGGAAGCTTTTTTAAAAAGGCTGCTCACGGTAGAGTCACAGAATTAATTAGCTTTTTGCTTTGCAAATATTTTTTTTATAACAGCTGGAAAATCACAAAAATAAATAGTATATATATTTGGCCAATAAAATCACTTTGATTTCAATAATAATCTAAATAACCAACCTAAAAGGCCTTTGATTAGCATGTTCCTTCAATGAAATGTACGTTGAGGTTTATTTTGATTCTCACAAGCACCAACCTGCTGCGTCAACAATTGAATTCAAATTTGTCCCAAAGGAATTCAAAGTAAATTTTTCTAGGCGATTTAATCTTTCCATTACTCTGATTTGTTTTAAAAATATAGAATAACTCAATCTCTATGATAAAACAATTACACATACATTCAGATTTTTATAGGACAAGATTGAAAACTTCTTACAAGTATGTATAAAACATCATCTGTATGTATAATTGTTTAACATGTAACAACTAGTCCTACTAATTGTGTTAAATTGTCATTTAATATCAATTGCTTGAGTTTATCATTATGTGTTTTGTTTTTTTTTACACAGTTGGCAAGCATGTTGGCAAGGCGGCCCTTACGTAAGGACTTCTACCATTTTACTGTATAATTTTGATAGTGTTATCACCAGTACTGTTTTTGACAACTTCTCTATTCCTGCTGACTCTCTCCATCCGACTCATCCGCAGTCATTACCTTGGCGATAAGCAGGAGCTCAACAAGCGTGCAGTCGATGAAGACCCAAATGTTATTGTTTTTGAATGAAGAAAT // NRC-05Winter Flounder WF3 (SEQ ID NO: 85)ATGAAGTTCACTGCCACCTTCCTGGTGCTGTCCCTGGTCGTCCTAATGGCTGAGCCTGGAGAGTGTTTCTTAGGAGCCCTTATCAAAGGGGCCATACATGGTAGAGTCAAGGAATTAATTAGATTTTTACATGTCAAATAATGTAGTAGAACATATATAAGTAGTCAATATATTTGACCAAGTAGAATCATTTTGATTTCAATAATAATCAAAATAACAATCTCCAGGCGATTTAATATTTGCAATAATTGGATTTTATAGAATACGGAACAACTGGATCTTAATGCTAAAATAATCCAACATACATTCTGATTTTGCCAGGCAAAATTAAACACTACTTTAAAGTATGTATAAAACATAATCTGTATGTTATAACAAATACTCCAAGCAATTGTGTGATGGAAATGTATTCATTGTCATTTAATATAATTTGCTTGAGTTTATCATCTTGTGTTTTTGTTTGTTTTTTCACAGGTGGCAGGTTTATCCATGGGTAAGGACTTCTACCATCATGACTGTGTATTTTTAATATTATTATCATCAGTACTGTTATTGACAACTTCACTTGTCTCGCTGACTCTCTCCATCAGAATGATCCAAAACCATCACGGTTATGACGAGCAGCAGGAGCTCAACAAGCGCGCAGTCGATGAA // NRC-06 Winter FlounderWF4 (SEQ ID NO: 86)GCCCACTTTGTATTCGCAAGGTAATATCAATATTTTTCAAATTCATTTAGACGAGACCAACCTTTTGGGAAATCTGCTCAGCTTATTACTGTATAATGCAAATGTTAATGATCTTTATTTTTCTGTTTTTTTTTTGTAGAATGAAGTTCACTGCCACCTTCCTCATGATGTTCATCTTCGTCCTCATGGTTGAACCTGGAGAGTGTGGTTGGGGAAGCATTTTTAAGCATGGTCGTCATGGTAAAGTCACGGAATTAATTAGCTTTTAACTTTGCAAATATTGTTTTTTTTTTTAACAGCTGGAAACTCACAAAAATAAATAGCCGATATATTTGGCCAATTATAATCACTTTGATCTAAATAACAACCTAAAAGGCCTTTGATTAGCATGTTTCTTCAATAAAATGATTGAACACTACTTAAAGGTATGTATAAAACATCATCATGTGTTTTTGTTTGTTTTTACACAGCTGCCAAGCATATTGGCCATGCAGCCGTTAAGTAAGGACTTCTACCATTATTACTGTATAATTTTGATAGTATTATCACCAGTATTGTTATTGACAACTTCTCTTTTTCCTGCTGATCCGACTCATCCGCAGTCATTACCTTGGCGAGCAGCAAGATCTCGACAAGCGCGCAGTCGATGAAGACCCAAATGTTATTGTTTTTGAATGAAGAAAT// NRC-07 Yellowtail Flounder YT2 (SEQ ID NO: 87)ATGAAGTTCACTGCCACCTTCCTCATGATGTGCATCTTCGTCCTCATGGTTGAACCTGGAGAGTGTCGTTGGGGGAAATGGTTTAAAAAGGCCACACACGGTAGAGTCACAGAATTAATTAGCTTTTTGCTTTGCAAATATTTTTTTATAACAGCTGGAAAATCACAAAAATAAATAGTCTATATATTTGGCCAATTAGAATCACTTTGCTTTCAATAAAAATCTAAATAACAACCTAAAAGTCCTTTGATTAGCATTTTCCATCAATGAAATGGACGTTGAGGTTTATTTTGATTCTCACATGCACCGACCTGGTATGTCAACAATTGAATACAAATTTGTCCCAGAGGAATTCAAAGGAAATTTTTCTAGGCGATCTAATCTTTCCATTACTCGGATTTGTTTTTAAATATATAGAATAACTCAATCTCTATGATAAAATAATAACACATACGTAAAGATTTTTACAAGACAAGATTGAAAACTTCTTAAAAGTACGTATAAAACATCATCTGTATTTATAATTGTTTAACATTTAACAAATAGCCCTACTAATTGTGTTATGGAAATGTATAAATTGTCATTTAACATAACTTGTTTGAGTTTATCATTATTTGTTTTTGTTTGTTTTTACACAGTTGGCAAGCATGTTGGCAAGGCGGCCCTTACGTAAGGACTTCTACCATCATTACTGTATAATTTTGATAGTATTATCACCAGTACTGTTATTGACAACTTCTCTTGTCCTGCTGACTCTCTCCATCCGACTCATCCATAGTGCTTACCTTGGCGACAAGCAAGAACTCGACAAGCGCGCAGTCGATGA //NRC-08 Winter Flounder WFX (SEQ ID NO: 88)TAATAAAACTAATGTGTAAAGTCTTCCACTTTTTTTACTGTATTTACTTAAACAGAAAATTATTCTCACGATTCTGGAGCTGCAGCCACTAAGTGTTGCTTCATGAAGTGAATACACAATTGTTCTAACAACCACTCACCCAATTAACCAGAATCTACAAAGTGAGGAAGTGAGAGGAGTCGTCCTGTGTTTTCAAATTTTTTGAATGATCTACCACTATGTGAGCTCCTCCTGTTATAGCTCTAAATGTTACACAATGAATGTGAAGTCAGTTCTGTGTATATAAAGAGTTGCCTCTGTAGAGCATACAACAGATTTCACCTTTGAATCTCACAAACCTCACTTTGTATTCGACAGGTAAGATCGATATTTTTCAAACTCATTTAGACGAGACCAAGTATTTGGGAAATGTGCTCAGCTTGTCAATGTATAATGCAAATGTTAACAATCGTTTTGTTCTTATGTTGTGTTTGTAGGATGAAGTTCGCTACTGCCTTCCTGATGTTGTCCATGGTCGTCCTCATGGCTGAACCTGGAGAGTGTCGTTCTACAGAGGACATCATCAAGTCTATCTCGGGTAGAGTCCAGGAATTAATTATTATCAATAACAATGAAATAACAACCAAAAGGCCTCTGATTAGCATGTTCCTTCAATGAAATGGTCGTTTTTTATCTATTTTGATTCTCACATGCAACGACCTGCTGCGGCAACATTTGAAAATCAATCTTTTTTACACAAATTCAAAGTACATTGATTTATTCGATTTAATCTTAACATTAATCAGATTTGTTTTTGTTTAAATATATCGAATAACTGGATCTCTATGATAAAATAATTAAACATACATTCTTATTTTACCAATCAAGATTGAACACTTCTTAAAAGTACGTATAAAACATCATCTGTATGTATAATTGTTTGATTGTTAAGTAATATTTCCAATAATTGTGTAATGGAAATGTATTAATTGTCATTTAATATAATTTGCTTGAATTTATCACCATGTGTTTTTTGTTTGTTTTTAAACAGGTGGAGGTTTTCTCAATGCGTAAGGACTTCTATCATCATTACTGTGTAATTTTTATAGTATTATCATCAGTACTGTTATTAACAGCTTCTCTTGTCTCACTGACTCTCTCCATCAGAATGAACGCCGGTTACAATGAGCAGCAGGAGCTCAACAAGCGCTCAGATGATGATGACAGCCCCAGTCTTATTGTTTTTGACTGAAGAAGTCGCCCTGAAGGAGCCTTCAGATGATATATTATGCTTCTTGCTCTTCATTGAAATAAATCAAAC // NRC-09 and NRC-b Winter Flounder WFY and WFZ (alternativesplice products from the same pseudogene) (SEQ ID NO: 89)GAGCTCGATCAAACCAGACAAAGTTGCCTTCCTTCACAACAATAGAGTGGAAGAGAAAACAGGAGAGGACTTGTATCCTCCTGATGCTGAGAAGAAGAAATAAGAAAGACTTGCAGCATTGATACTTTTACTTATACAGAAAACCTATAAACATGACGGGAGCATAAGTTAAAGTCACAATACAGAAGAGAACCAGAAGCCAAACTGCAGCAAATTTACTGGTATTCATATGATACTGGAGCCAAAGCAACGCAGAGACTCAGCAGCAGTGAACCAAAGAGTTTAACTGTACTTGTGTCCAGGTTGAATGAAAGTATTGAATAAAAAAAACCAAGACAGAACATGCATATTTTTTTGGAATGGAATATAAGTCAGGAGAATATGTGTTGTTGTGGTGGCAGGATCCATCACTCTGTCAAGTTAACACAAGAACTTTTAGAAACATAGATACGATCTCAAGTAAACTTCCATTTACTATTTGACTTTTTTTAAATACTTACAAATTATATTTTAAAAAGCAACAATAAATCAGAGATAACTTCATGGAGAAGTCTATATTCATATTTGTGAGCTGAACATTCATGCTGCCTGTTCTATCACATCTGAGTGTGGAGGCCACTGACGTTTACTGACCTCAACGTCTACCGCTCTAATGCATTTGGAGTTAAAGGTAAGCATTTTGTTATTTGTCTTCACTGTATTGATACTAAATATACAGGGTTACAAATACAGTTAAAACAAGAGAGACGAGGTGTCGAAAGCTTCAGCATCAATGTGCTGATCGCTGATAGCTGATCTTACCCGACACCGGTGACATGGCATCAAAATGACCACCTCTTTTTTCTTCTCTTTTTTTTGTAGGACGAAGTTCGCTGCCGCCTTCCTCGTGTTGTTCATGGTCATCGTCATGTTTGAACCTGGAGAGTGTTTTTTTAGATTGCTTTTTCACGGGGTCCACCATGGTAGGGTCCCGGAAGTAATTTGATTATTACATGCCAAATATTTTAATGAAACATACCTTATGAGTAGTTGTATTATTTGGACAAGTAGAATCTCTATGATTTCAGTAGTAATTAGAATAACAATCAAAAAGGCCTTTGATTAGCATGTTTCTTCAATGAAATGGACATTGAGGTTTATTTTGATTCTCACATGCTACAGCAACAATTGAAATCAAATTTTTCGCAGAAGAAACTTAATTAACATTGTTGTGCAATAGTGCTTAAAAAGTGTTACCATGGAATGGTGTGCGTTTAGGCACTCAATAAATTTTGGTTATCAAATTAAATTAAAAAAATTAATATTTAAAATATTAATATTAAATCATAACTTTAATTGTTTAAAGTTCTCGCGGGGAACCACCCTTCTTCTGAAGGTAAAGGATAGCCAATTTATTGATTAAGATCAGTCTCATTTAGATCTAGTTCAAATAGAAATCTCAATATTTTACCATCGAAGATTTTATAATGAACAGTGAAGGTTATGGAGTTCTAAACAGTGTAACAGTTGGCAAAGTTCACTATTGCAATATTAATGACAGACCATTTGTGAAAGAAGAACATTTATTATGAGCATAATAAAGTATGAAAGCACGAATTACTAAACAATCAAAGCTAACTAACAAGGACGTGTGTGGGTGTGTGTGTGAATGTAAATAAGGGGGGGGCTCAAACTGGTGGCCTACAAGAAGAGCCTTAAGATAGCAACCACAAGGGCTGTACCATAAATGTTGTAGTAAAAAGAGTTATTAAAATGAGTTAGAATAACTAATGACTAATTAGTAGACAAACTAGTAGACAAACTAAACAACTAACAATAACAAGGAAGTGTGTGTGAGTGTGTTTGTGTGTAAATGTTAATTAGGGGCTCTCAAACTGGTGTCTTACCAGAAGAGTAAGATAACAATTCCCCCCCTTCTTCTGAGGTTGTTTTACGACTGTTGCTTTATGGCCGTGAGGGAAGGTTTAACTCGGTGACATGCTATACGTGTCTGTGTAGATGTTAATCAGAGAATGCCAGAGTCAGAGAGACCTACGGAGGAAGTCTGTGAAGGGCCTATCTAACATTAGCTTTCCTTTAACTTATAACACAATATCAGAAACACATATCAACCTTATAAACACACACAGAATCAAATAAACAGTCTTGCTTAGCATGTATAATTATTAAGCCCAGATTATGTTACCAGTCCGAGGGAAAGAGTTCAGTTGCAGTTCTGTGACGTCTCCTGGCTTTGTGGTCGTAGAGTTCTGCATTCGCGATTCTGTCGAGCCGTGTGCTCAGATGCAGGTTGAAGTTCTCCTGCAGGACATCGCGTCGCTGCGAGGATTTTGTAGAGCTTGAAGGGCGAGGAGATTTCCTTGAGTGGTGAGCTGGAAGCTGGACCTCTGACCTCTGGTTGTTGGTTGGAAGAGAAGAAAGCTGGAGCGGCGTGGTTTCTCCCTCTAGCCGATGCAGGAGGAGAAGCCGGCAGCCCCACTCCTTGAAGAGTTGTGGAGAGAGATGGGAGCAAAGAGCTAGATTTTGGGGAGACCTCTCCTTATATTGGCCCCGATGACCTCACAGGCCTTGGAACGGAGTGACCAATAGGAGTTGACCCTGGTAATTCTTGACACCTTTGTGGGACATTGTCAAGACCCCAGGACATGCAGCATCCTGTTACAATCTGGGAGACGGAGTTCCTTGACTGTCTCAGAACAATGAGAACCTGTGGCATCTTGGGGGATTGAGTCCACTCGAGCACATGCGGCATGTTTGTTCCAAGTTTGACTGAAAGGAGGCCTGTGGTTTGCACAAAAACCATGTCCCAACAACATTTTCTAGGCGATTTAATCTTTACATAAATTGGATTTGTTTTAAAAAATATATAGAATAACTCGATCTTTCTGCGTAAATAATAAAAAATAAATTCAAATTTGACCAGTCAAGATTGAACACTAATGAAAAGTACCTATAAAACATAATCTGTATGTATAGTTGTTTGACTGTTAAATAGTAGTCCTAACAATTGTGTAATGGAAATGTATTCATTGTCTTTTAATACTATTTGCTTATCATAATGTGTTTGTTTGTTTTTTAGCAGGTGGAGGTTATCTCAATGCGTAAGGACTTCTACCATCATTACTGTGTAATTGTATTAGTTTTATCATCAGTACTGTTATTGACAACGTCTCTTGTCTTGCTGACTTGACTCTCTTCATCAGATTAAACCCAGGGCCGGTTACAATGAGCAGCAGGAGCTCGACAAGCGCGCAGTCGATGACAACCTCAGTGCTATTGTTTTTTACTGAAGAAGTCGACCTGAAGAATCTTTTGAAATGATATGAAATGTTTGCCTTTCAATGAAATAAATCAAACATGACTGGATATTTGTTCTTTTGCATTGATGTATTGTTGAGTGACAGTTGAATAATTTTGGAAAACTTATAACAGATCTCAATTTTAGGATGTCAAATCATTTCTCTGTGTCTTATTCAAATATGAGATTTAACAATGACAAT // NRC-11 American Plaice AP1 (SEQ ID NO: 90)GCCCACTTTGTATTCGCAAGGTAAGATCAATATTTTTCAAATTCATTTAGACGAGACCAACCGTTTGCGAAATGTGCTCAGCTTGTTATTGTATAATAACAAAGTTAACGATCTTTATTTTTCTGTTTTTTTGTAGAATGAAGTTCACTGCCACCTTCCTGATGTTGTTCATCTTCGTCCTCATGGTTGAACCTGGAGAGTGTGGATGGAAAAGTGTGTTTCGTAAGGCTAAGAAAGGTAGAGTCACGGAATTAATTAGCTTTTTACATTGCAAATAGATTTTTTATAACAGCTGGAAAATCACAAAAATAAATAGTCGATATATTTGGCCAATTAGAATCACTTTAATTTCAATAATAATCTAAATAACAACCTAAAAGGCCTTTGATTAGCATGTTTCTTCAATGAAATGGACATTGAGGTTTATTTTGATTCTCACATGCACCGACCTGTGCGGCAACCATTGAATTCAGATTTGTCCCAGAAGAATTCAAAGTACATTTTTCCAGGCGATTAAATCTTTCCATTACTCAGATTCAAAAATAAATAAATGGAATAATTGAAGCACTATGATAAAATAATTACACATTCACTCTGACTTTACAAGTCAAGATTGAACACTATTAAAAAGTGTGTATAAAACAACATCTGTATGCATAATTGTTTAACTGTTAATAGTCCTAATAATTGTTTTATGGAAATGTATTAATTTACATTTAATATTATTTGCTTGAGTTTACCATCATGTGTTTTTGTTTGTTTTTACACAGTTGGCAAGACTGTTGGCGGCTTGGCCCTTGAGTAAGGACTTCTACCATCATTACTGTATAATTTTGATAGTATTATCACCAGTACTGTTATTAACTACTTCTCTTGTCTGCTGACTCTCTCCATCCGACTCATCTGCAGTCATTACCTTGGCGAGCAGCAGGAGCTTGACAGCGCGCAGTCGATGAGGACCCCAGTGCTATTGTCTTTGACTGAAGAAGTCGCCTTGAAGGAG // NRC-12 American Plaice AP2 (SEQ ID NO: 91)ACTTTGTATTCGCAAGGTAAGATCAATATTTTTCAAATTCATTTAGACGAGACCAACCGTTGGCGAAATGTGCTCAACTTGTTATTGTATAATAACAAAGTTAACGATCTTTATTTTTCTGTTTTTTTGTAGAATGAAGTTCACTGCCACCTTCCTGATGTTGTTCATCTTCGTCCTCATGGTTGAACCTGGAGAGTGTGGATGGAAAAAATGGTTTAATAGGGCTAAGAAAGGTAGAGTCACGGAATTAATTAGCTTTTTACATTGCAAATAGATTTTTTATAACAGCTGGAAAATCACAAAAATAAATAGTCGATATATTTGGCCAATTAGAATCACTTTAATTTCAATAATCTAAATAACAACCTAAAAGGCCTTTGATTAGCATGTTTCTTCAATGAAATGGACATTGAGGTTTATTTTGATTCTCACATGCACCGACCTGTGCGGCAACCATTGAATTCAGATTTGTCCCAGAAGAATTCAAAGTACATTTTTCCAGGCGATTAAATCTTTCCATTACTCAGATTCAAAAATAAATAAATAGAATAATTGAAGCACTATGATAAAATAATTACACATTCACTCTGATTTTACAAGTCAAGATTGAACACTATTAAAAACTGTGTATAGAACATCATCTGTATGTGTAATTGTTTAACTGTTAATAGTCCTAATAATTGTTTTATGGAAATGTATTAATTTACATTTAATATTATTTGCTTGAGTTTACCATCATGTGGTTTTGTTTGTTTTTACACAGTTGGCAAGACTGTTGGCGGCTTGGCCGTTGAGTAAGGACTTCTACCATCATTACTGTATAATTTTGATAGTATTATCACCAGTACTGTTATTAACTACTTCTCTTGTCTCGCTGACTCTCTCCATCCGACTCCTCTGCAGTCATTACCTTGGCAAGCAGCCGGAGCTCGACAAGCGCGCAGTCGATGAGGACCCCAGTGCTATTGTCTTTGACTGAAGAAGTCGCCTTGAAGGAGCCTTCAGAA // NRC-13 American Plaice AP3 (SEQ ID NO: 92)TTGCCCACTTTGTATTCGCAAGGTAAGATCAATATTTTTCAAATTCATTTAGACGAGACCAACCATTTGGGAAATGTGCTCAGCTTGTTACTGTATAATGCAAAAGTTAAGTATCTTTATTTTTCTGTTTTTTTTTGTAGAATGAAGTTCACTGCCAACTTCCTCATGTTGTTCATCTTCGTCCTCATGTTTGAACCTGGAGAGTGTGGTTGGCGAACATTGCTTAAAAAAGCTGGTCACGGAATTAATACGCTTTTTACATTGCAAATAGATTTTTTATAACAGCTGGAAAATGACAAAAATAAATAGTCGATATATTTGGCCAATTAGAATTATTTTGATTTCAATAATAATCTAAATAACAACCTAAAAGGTCTTTGATTAGCATGTTTCTTCAATGAAATGGACATTGAGGTTTATTTTGATTCTCACATGACCGACCTGCTGCGGCAACAATTGAATTCAGATTTGTCCCAGAAGAATTCAAAGTAAATTTTCCAGGGGATTAAATCTTTCCATTACTCGGATTTAAAAAAAAAAAAAATAGAATAACTGAATTGCCATGAAAAAATAATTACACATACTGTCTGATTTTACAAGTCAAGATTGAACACTACTTAAAAGTATGTATAAAACATCATCTGTATGTATAATTGTTTAACTGTTAACAAATAGTCCAAATAATTGTGTTATGGAAATGTATTAATTGTCATTAAATATAATTTGCTTGAGTTTATCATCATGTGTTTTTTTTTTTTTTTTACACAGAGGTTAAGACTGTTGGCAAGTTGGCCCTTAAGTAAGGACTTCTACCATCATTACTGTATAATTTTGATAGTATTATCACCAGTACTGTAGTACTGACAACTTCTCTCTCCACCCAACTCATCCGCAGACATTACCTTGGCAAGCAGCCGGAGCTCGACAAGCGCGCAATTGATGACGACCCCAGTATTATTGTTTTTGACTGAAGAAGTCGCCTTGAAGGAGCCTTCAGAA // NRC-14 Witch Flounder GcSc4C5 (SEQ ID NO: 93)ATGAAGTTCACTGCCACCTTCCTCATGATGTTCATGGTCGTCCTCATGGCTGAACCCGGAGAGGCTGGTTGGGGAAGTATTTTCAAACATATTTTCAAAGCTGGAAAGTTCATCCATGGTGCGATCCAGGCACACAATGACGGCGAGGAGCAGGATCTCGACAAGCGCGCAGTCGATGA // NRC-15 Witch Flounder GcSc4B7 (SEQ ID NO:94)ATGAAGTTCACTGCCACCTTCCTGGTGTTGTTCATGGTCGTCCTCATGGCTGAACCTGGAGAGGGTTTTTGGGGAAAGCTTTTGAAATTGGGCATGCATGGAATCGGGCTGCTCCATCAGCATTTGGGTGCTGACGAGCAGCAGGAGCTCGACGAGCGCTCAGAGGAGGACGAGCCCAATGTTATTGTTTTTGAATGAAGAAGTCGCATTGAAGGAGCCTTCAG// NRC-16 and NRC-17 Witch Flounder GC3.8 (SEQ ID NO: 95)ATGAAGTTCACTGCCACCTTCCTGGTGTTGTTCATGGTCGTCCTCATGGCTGGATCCGGAGAGTGTGGTTGGAAAAAGTGGCTCCGTAAAGGTAGAGTCATGGATTTAATTTGCTTTTTACATTGCAAATACTTTAATATAACATAGTTGGAAAACCACAAAAATAAGTAGTCGATATATTTGGCCATATAGAATCACTTTGATTTCAATAATAATCAAAACAACAATCAAAAAGCCCATTGATTAGCATGTCCCTTCACTAAAATGGACATTGTAATTTATTTTGATTCTCACAGGCACCAACCTGCTGCGGCAACAATTGAAATCAAATTTGTCTCAGAAGAATTCAAAGTACATTGTTCTAGGCGATTTAATCTTTCCATTCATCGGATCTGTTTTTAAAAATATAGAATAACTGGATCTCTATGTTAAAATAATAAAACACACATTCTGATTTTACCTGTCAAGATTGAACACGACTTAAAAGTATGTATAAAACATCATCTGTATGTATAATTGTTTAACTGTCAACTAATAGTCCAAATAATTGTGTTATGGAAATGTATTCATTGTCATATAATATCATTTGCTTGAATTTATCACCATGTGTTTTTGTTTGTTTTTACACAGGTGCCAAGCACCTTGGCCAGGCGGCCATTAAGTAAGGACTTCTACCATCATTACTGTGTAATTTTAACAGTATTATCATCAGTACTGTTATTGACAACTACTCTTGTCTCTGTTACTCTCTCCAGGGGTTTGGCCTCTTGCGAAGAGCAGCAGGAGCTCGACAAGCGCTCAATGGATGACGAGCCCAGTGCTATTGTTTTTGACTGAAGAAGTCGCCTTGAAGGAGCCTTCA // NRC-18 Witch Flounder GC3.2 (SEQ ID NO:96)ATGAAGTTCACTGCCACCTTCCTGGTGTTGTTCATGGTCGTCCTCATGGCTGGATCCGGAGAGTGTGGTTGGAAAAAGTGGTTCACTAAAGGTAGAGTCATGGATTTAATTTGCTTTTTACATTGCAAATACTTTAATATAACATAGCTGGAAAATCACAAAAATAAGTAGTCGATATATTTGGCCATATAGAATCACTTTGATTTCAATAATAATCAAAACAATAATCAAAAAGCCTATTGATTAGCATGTTCCTTCACTAAAATGGACATTGTAATTTATTTTGATTCTCACAGGCACCAACCTGCTGTGGCAACAATTGAAATCAAATTTGTCTCAGAAGAATTCAAAGTACATTGTTCTAGGCGATTTAATCTTTCCATTCATCGGATTTGTTTTCAAAAATATAGAATAACTGGATCTCTATGTTAAAATAATAAAACACATTCTGATTTTATCTGTCAAGATTGAACACGACTTAAAAGTATGAATAAAACATCATCTGTATGTATAATTTTTTAACTGTCAACTAATAGTCCAAATAATTGTGTTATGGAAATGTATTCATTGTCATATAATATCATTTGCTTGAATTTATCACCATGTGTCTTTGTTTGTTTTTACACAGGTGAAAGGTTATCCCAGAGGTAAGGACTTCTACCATCATTACTGTATAATTTTAATAGTATTATCATCAGTACTGTTATTGATAACTTCTCTTGTCTCGCTGACTCTCTCCATCAGGCATTTCGCTGACGTCGAGCAGCAGGAGCTCGACAAGCGCTCAGTGGATGACGAGCCCAGTTCTATTGCTTTTGACTGAAGAAGTCGCCTTGAAGGAGCCTTCAG // NRC-19 Halibut HB26 (SEQ ID NO: 97)TTATGAAGTTCACTGCCACCTTCCTGGTGTTGTTCATGGTCGTCCTCATGGCTGAGCCTGGAGAGTGTTTTTTGGGATTGCTTTTTCACGGGGTCCACCATGGTAGGGTCACGGAAGTAATTCGATTTTTACATGGCAAATATTTTAAGATAACACACCATATGAGTAGTCGATATATTTGACCAATTAGAATCACTTTAATTTCAATAATAATCACAATAACAATCTCTAGGCCATTTAATCTTTCCATTAATCGGATTTGTTTTTTTAAATATAGAATAACTGGATCTCTATGTTAAAATAATAAAACATACATTCTGATTTTACCAGTCAAGATTGTACGCTACTTAAAAGTATGTATAAAACATCATCTGTATGTATAATTGTTTAACTGTTAACTAATAGTCCAAATAATTGTGTAATGGAAATGTATTAATTGTCATTTAATATCATTTGCTTGAATTTATCACCATGTGTTTTTGTTTGTTTTTACACAGTTGGAAAGTGGATCCATGGGTAAGGACTTCTACCATCATTACTGTGTATTTTTAATAGTATTATCATCAGTACTGTTATTGATATTTTCTCTTGTCTCGCTGACTCTCTCCATCAGACTCATCCATGGGCATCACGGTTACGACGAGCAGCAGGAGCTCGACAAGCGCGCAGTCGATGAAA// NRC-20 Halibut HB18 (SEQ ID NO: 98)TTATGAAGTTCACTGCCACCTTCCTGGTGTTGTTCATGGTCGTCCTCATGGCTGAACCTGGAGAGGGTTTTTTGGGAATTCTTTTTCACGGGGTCCACCATGGTAGAGTCACGGAATTAATTCGATTTTTACATGGCAAATATTTTAAGATAACACACCATATGAGTAGTCGATATATTTGACCAATTAGATTCACTTTAATTTCAATAATAATCACAATAACAATCTCTAGGCCATTTAATCTTTCCATTAATCGGATTTGTTTTTTTAAATATAGAATAACTGGATCTCTATGTTAAAATAATAAAACATACATTCTGATTTTACCAGTCAAGATTGAACACTACTTAAAAGTATGTATAAAACATCATCTGTATGTATAATTGTTTAACTGTTAACAATAGTCCAAATAATTGTGTTATGGAAATGTATTAATTGTCATTTAATATCATTTGCTTGAATTTATCACCATGAGTTTTTTGTTTGTTTTTACACAGGTAGAAAGAAGGCCTTGCAGTAAGGACTTCTACCATCATTACTTTGTAATTTTTATAGTATTATCATCAGTACTGTTATTGACAACTTCTCTTGTCTCGCTGACTCTCTCCATCAGGATGAACTCAGAGCGTCGCAGTTACGACGAGCGGCAGCAGCAGCAGCAGGAGCTCGACAAGCGCGCAGTCGATGAAA // NRC-101 Yellowtail Flounder YT1 (SEQ ID NO: 99)GCCCACTTTGTATTCGCAAGGTAAGATCGATATTTTTCAAACTCATTTAGACGAGACCAAGCATTTGTTGAAATGTGATAAGCTTCTAACTTTATAATGCAAATGTTAACAATCTTITTGTTCTGTTGTTTTTGTAGGATGAAGTTGGCTGCCGCCTTCCTGGTGCTGTTCCTGGTCGTCCTCATGGCTGAACCTGGAGAGGGTTTCTTGGGATTTCTTTTTCACGGTATCCACCATGGTAAAGTCACTCATTTAATACATTTTTACATGGCAAATATTTGAATATAACATACTATATGAGTTGTCAATATATGTGGCCAAGTAGAAGCACTTTGATTTCAATAATAATCAAAATAACAATCACTAAGCCATTTAATAATTGAATTAATTACATTTGTTTTAAAAAAATATAGAATAACTGGATCTTTATGCTAAAATAATTAAACCTAAATTCAGATTTTACCACTCAAGATTGAACACTACTTAAAAGTATGTAAAAAAAACATCATCTGTATGTATAATTAAATACTAGTCCAGTTAATTGTTTTATGGAAATGTGTTAATTGACATATATCATTTGCTTGAACTTATAATGTGCTTTGTTTGTTTTTACACAGGTATCAGGGCGATCCATCAGTAAGGACTTCTACCATCATGACTGTGTATTTTTAATAGTATTATCATCAGTACTTTTATTAACAACTTCTCTTGTCTCGCTGACTCTCTCCATCAGTCTCATCCATGGTCAAAGATACGACGAGCAGCAGGAGCTTGACAAGCGCTCAGTCGATGACAACCCCGGTGCTATTGTTTTTGACTGAAGACGTCGCCTTGAAGGAGCCTTCAG // NRC-102 Yellowtail Flounder YT3 (SEQ ID NO: 100)ATGAAGTTCACTGCCACCTTCCTGGTGTTGTCCATGGTCGTCCTCATGGCTGAACCTGGAGAGGGTTTCTTTGGAGCCCTTATCAAAGGGGCCATCCATGGTGGCAAGTTGCTCCATAAACTCATCAAAAAAAAACATGAACATCACGGTTATGGCAAGCATTGGGGGCTTGACAAGCGCGCAGTCGATGA // MRC-103 Winter Flounder WF-YT(SEQ ID NO: 101)TTGAAAGTGAGGAAGTGAGAGGAGGACTAGGTCCTGTGTTTTCAGTCGTTGAATTATCTAACACTATCTGAGCCCCTCCTGCAATAACTCTAAATGTTACACAGTGACTAGGAAGTCAGTCCTGTGTATATAAAGAGTTGCATCTGTTGTTATCAGTAGACAACAGATTACACCTTTGAATCTCACAAAGCTCATTTTGTATTCGACAGGTAAGATCGATATGTTTCAAACTCATTTAGATGAGACCAAGCATTTGGGAAATGTGCTCAGCTTCTAACTGTATGATGCAAATGTTAACAATCTTTTTGTTCTGTTGTTTTGTAGGATGAAGTTGGCTGCCGCCTTCCTGGTGCTGTTCCTGGTCGTCCTCATGGCTGAACCTGGAGAGAGTTTTTTGGGATTTCTTTTTCATGGTATCCGCCATGGTAGGGTCACTGAATTGATACATTTTTACATGGCAAATATTTGAATGTAACATACTATATGAGTTGTCAATATATGTGGCCAAGTAGAAGCACTTTGATTTCAGTAATAATCAAAATAACAATCACTAGGCCATTTAATAATTGCATTAATTACACTTGTTTTTATATAGAATATAGAATAACTGGATCTTTATGCTAAAATTAATAAACATGAATTCAGATTTTAAGATTTTTCAAGATTGAAAACTACTTAAAAGTATGTAAAAAAACATCATCTGTATGTATAATTAAATACTTGTCCAGATAATTGTGTTGTGGAAATGTGTTAATTGACATATATCATTTGCTTGAATTTATCATTATCTGCTTTGTTTGTTTTTACACAGGTATCAAGGCGATCCATGGGTAAGGACTTCTACCTTCATGACTGTGTATTTTTAATAGTATTATATTCAGTACTGTTATTGAAAACTTCTCTTGTCTCGCTGACTCTCTCCATCAGAATGATCCATGGTAACAGTTTAGACGAGATGCAGGAGCTCGACAAGCGCTCATTCGATGACAACCCCAACGCAATTGTTTTTGACTGAAGAAGTCGCCCTGAAGGAGCCTTCAGATGATATATAATGCTTCTTGCTTTTCAATGAAATAAATTGAATAATTACCCGCAACAGC // NRC-104 Winter Flounder WF1-like(SEQ ID NO: 102)TACTTTTATCTACCACTATGTGAGCTCCTCCTGTTATAACTCTAAATGTTACACAATGAAGATGAGGTCAATTCTGTGTATATAAAGAGTTGCCTCTGTATAGTAGACAACATATTTCACCTTTGAATCCCACAAAGCTCACTTTGTACTCAACAGGTAAGATCGATATTTAAAAACTAATTTAGACGAAACCAAGCATTTTGGGGAATTTGCTCAACTTCTAAATGTATGATACAAATGTTAACAATCTTTTATTTCTGTTGTTGTTTTTTGTAGGATGAAGTTCACTGCCACCCTCCTCCTGTTGTTCATCTTCGTCCTCATGGTTGATCTCGGAGAGGGTCGTCGTAAGAAAAAGGGGTCGAAGAGAAAGGGGTCCAAGGGAAAGGGGTCCAAGGGAAAGGGCAGGTGGTTGGACAGGATTGGTAAAGGTAGAGTCACGGAATTAATTTGCTTTTTACATTGCAAATATTTTTCATATAACATTGCTGGAAAATCACAAAAATAAGTAGTCAATATATTTGGCCAAATAGAATCACTTTGATTTCAATAATAATCAAAATAACAACCTAAAAGGCCTTTGATTAGCATGTTCCTTCAATGAAATGGACATTGTAATTTACTTTGATTCTCACATGCTACGACCTGCTGCAGCAACATTTGAAAATAAATTTGTCCCAGAAGATTTTAAAGTACATTGTTATAGGCGATTTATCTTTCTATTACTCAGATATTTGTTCAAACCAATAGAATAACTGGATCTCTATGCTAAAATAATAAAACACACATTCAGATGTTACCAGTCAAGATTGAACGCTGTTTAAAAGTAAGTATGAAACATCCTCTGTATGTATAATTGTTTAACTGGTAACTTATAGTCCTAATAATTGCGTTATGGAAATGTATTAATTGTCATTTAATATAATTTGCTGGAATTTATCACTGTGTGTTTTTGTTTGTTTTTACACAGCTGGCGGGATAATTATCGGGGGGGCCCTTGAGTAAGGACTTCTACCATCATTACTGTGTAATATTTATAGTTATGATCAGTACAGTTATTAACAACTTCTCTTGTCTCGCTGAACTTCTCCATCAGTCACCTCGGGCAGGGGCAGGTGCAGGGGCCGGATTACGACTACCAGGAGGGGGAGGAGCTCAACAAGCGCTCAGACGATGATGACAGCCCCAGTCTTATTTTTTTTGACTGAAGAAGTCGCCCTGAAGGAGCCTTCAGATGATATATAATGCTTCTGGCTTTTCATTGAAATAAATAATACGTTTACCTGCAACAGCAACCATG // NRC-105 Halibut Hb29 (SEQ ID NO: 103)TTATGAAGTTCACTGCCACCTTCCTGGTGTTGTTCATGGTCGTCCTCATGGCTGAACCTGGAGAGGGTTTGGGAAATTGGATGGGGCCCCATATCAGCGGTAGAGTCACGGAATTAATTTGCTTTTTCCATTGCAAATATTTTAATATTGCATAGCTGGAAAATCACGAAATAAGTAGTCGATATATTTGGCCAAATAGAATCACTTTGATTTCAATAATAATCAAAATAACAATCAAAAAGGCCTTTGATTAGCATGTTCCTTCAATAAAATGGACATTGAAGTTTATTTTGATGCTCACATGCACCGACCTGCTGCGGCAACAATTGAAATCAAATTTGTCTCAGAATTTAAAGTACATTTTTCTAGGTGATTTAATCTTTCCATTAACTTGATTTGTTTTTATAAATATAGAATAACTGGATCTTTATGCCAAAATAATAAAACACACATTCTGATTTTACCAGTCAAGATTGAACACTACTTAAAAGTAATATAAAACATCATCTGTATGTATAATTGTTTAACTGTTAACAAAAGTCCAAATAATTGTGTTATGGAAATGTATTAATTGTCATTTAATATCATTTGCTTGAATTCATCACCATGTGTTTTTTGTTTGTTTTTACACAGGTGAAAAGAAGGCCTTGCAGTAAGGACTTCTACCATCATTACTTTGTAATTTTTATAGTATTATCATCAGTACTGTTATTGACAACTTCTCTTGTCTCGCTGACTCTCTCCATCAGGATGAACTCAGAGCGTCGCAGTTACGACGAGCGGCAGCAGCAGCAGCAGGAGCTCGACAAGCGCGCAGTCGATGA //NRC-106 Halibut HbSc1A13 (SEQ ID NO: 104)ATGAAGTTCACTGCCACCTTCCTGGTGTTGTTCATGGTCGTCCTCATGGCTGAACCTGGAGAGGGTTTGGGAAATTGGATCGTGCGCCCTATCGGAGGTGAAAAGAAGGCCTTGCAGATGAACTCAGAGCGTCGCAGTTACGACGAGCGGCAGCAGCAGCAGCAGGAGCTCGACAAGCGCGCAGTCGATGAAA // NRC-107 Halibut HbSc1A24(SEQ ID NO: 105)ATGAAGTTCACTGCCACCTTCCTGGTGTTGTTCATGGTCGTCCTCATAGCTGAACCTGGAGAGAGTCTTTTTGGAAAGTTCCTCAAGAAAGTTGTCCATGCTGGCACGTCAATTGGCGAGACAGCCTTGCATGTCGCCGCAGAGCATCACGGGCTTCATGCGCATCACGGGTGTCACGGGCGTCACGGGGGTCACAGGCGTCACGGGGGTCACAGGCGTCACGGGCGTCGCGGTTACGACGAGCAGCAGCAGGAGGAGCTCGACAAGCGCGCATTCGATGA // NRC-108 HalibutHbSc1B34 (SEQ ID NO: 106)TATGAAGTTCACTGCCACCTTCCTGGTGTTGTTCATGGTCGTCCTCATGGCTGAACCTGGAGAGGGTTTGGGAAATTGGATGGGGCCCCATATCAGCGGTAGAAAGAAGGCCTTGCACATGAACTCAGAGCGTCGCAGTTACGACGAGCGGCAGCAGCAGCAGCAGGAGCTCGACAAGCGCGCAGTCGATGAAA // NRC-109 Halibut Hb17 (SEQID NO: 107)ATGAAGTTCACTGCCACCTTCCTGGTGTTGTTCATGGTCGTCCTCATGGCTGAACCTGGAGAGTGTTTTTTGGGATTGCTTTTTCACGGGGTCCACCATGGTAGGGTCACGGAAGTAATTCGATTTTTACATGGCAAATATTTTAAGATAACACACCATATGAGTAGTCGATATATTTGGCCAATTAGAATCACTTTGATTTCAATAATAATCAAAATAACAATCTCTAGGCGATTTAATATTTGCATTAATTGGATTTGTTTTTAAAAATATAGAATAACTGGATCTTTATGGTAAAATAATTAAACATACATTCTGATTTTACCAGTCAAGATTGAACACTACTTAGAAGTATGTATAAAACATCATCTGTATGTATAATTGTTTAACTGTTAACGAATAGTCCAAATAATTGTGTTATGGAAATGTATTAATTGTCATTTAATATCATTTGCTTGAATTTATCACCATGTGTTTTTGTTTGTTTTTACACAGTTGGAAAGTTGATCCATGGGTAAGGACTTCTACCATCATTACTGTGTATTTTTAATAGTATTATCATCAGTACTATTATTGACAACTTCTCTTGTCTCGCTGACTCTCTCCATCAGACTCATCCATGGCGGTTACGACGAGCAGCAGGAGCTCGACAAGCGCGCAGTCGATGAA // NRC-110Witch Flounder GC1.2 (SEQ ID NO: 108)GCCCACTTTGTATTCGCAAGGTAAGAGCGATATATTTCAAATTCATTCGGATGAGACCAAGCATTTGGGAAATGTGCTCAGCTTGTTACTGTTTAATGCAAATGTTAACAATATCCTTTTTCTGTTGTTTTTGTAGAATGAAGTTCGCTGCCGCCTTCCTCATGATGTTCATGGTCGTCCTCATGGCTGAACCCGGAGAGGCTCGTTGGGGAACGTTCTTCAAACATATTTTCAAAGGTAGAGTCACAGAATTAATTTGCTTTTTACATTGCAAATATTTTCATATAACATAGCTGGAAAATCACAAAAATAAGGGCTTGATATATTTGGCAAAGTAGAATCCCTTTGATTTCAATAATAATCAAAATAAAAATCAGAAAGGCCTTTGATTAGCATGTTCCTTCAATAAAATGGACATTGTAGTTTATTTTGATTCTCAAATGCACCAACCTGCTGCGGCAACAATTGAAATCAAATTTGTCTCCGAAACATTTAAAGTACATTTTTCGAGGCAATTTAATCTTTCCTTTGATCGAATTCGTTTTTAAAAATATAGAATAACTGGATCTTTATGCTAAAATAATAAATCATACATTCTGATTTTACCAGTCAAGATTGAACGCTACTTAAAAGTATGTATAAAACATCATCTGTATGTATAATTGTTTAACTTTTAACTAATAGTCCTAATAATTGTGTTATGGAAATGTATTCATTGTCATTTAATATCATTTGCTTGAATTTATCACCATGTGTTTTTGTTTGTTTTTACACAGCTGGAAGGTTCATCCATGGGTAAGGACTTCTACCATCATTACTGTGTATTTTTAATAGTATTATCATCAGTACTGTTATTGATAACTTCTCTTGTCTCGCTGACTCTCTCCATCAGTGCGATCCAGGCACACAATGACGGCGAGCAGCAGGATCTCGACAAGCGCTCAGTGGATGATGAGCCCAGTGTTATTGTTTTTGAATGAAGAAGTCGCCTTGAAGGAGCCTTCAG // NRC-111 Witch Flounder GC1.3 (SEQ ID NO: 109)GCCCACTTTGTATTCGCAAGGTAAGAGCAATATATTTCAAATTCATTTAGACGAGACCAAGCATTTGGGATCTGTGCTCAACTTGTAACTGTATAATGCAAATGTTAACAATATTCTTTTTCTGTTGTTTTTGTAGAATGAAGTTCGCTGCCGCCTTCCTCATGATGTTCATGGTCGTCCTCATGGCTGAACCCGGAGAGGGTGCTTGGATACCTGCCTTGAATAGGATCTATCATGGTAGAGTCACAGAGTTAATTTGCTTTTTACATTGCAAATATTTTAATATAACATGGCTGGAAAATCACAAAAATGAGTACTCGATATATTTGGCAAAGTAGAATCCCTTTGATTTCAATAATAATCAAAAACACAATCAAAAAGGCCATTGATTAGCATGTTCCTTCAATGAAATGGACATTGTAGTTTATTTTGATTCTGACATGCACCAACTTGCTGCGGCAACAATTGAATTCAAATTTGTCTCAGAAAAATTTAAAGTACATTTTTCTTTCCATTAGTCGGATTTGTTTTAAAAAATACAGAATAACTGGATCTTTATGCTAAAATAATAAATCATACATTCTGATTTTACCAGTCAAGATTGAACGCTACTTAAAAGTATGTATAAAACATCATCTGTATTGATAATTGTTTAACTTTTAACTAATAGTCCTAATAATTGTGTTATGGAAATGTATTCATTGTCATTTAATATCATTTGCTTGAATTTATCACCATGTGTTTTTGTTTGTTTTTACACAGCTCTACTGAGGATCAATCGGTAAGGACTTCTACCATCATTACTGTGTAATTTTAATAGTATTATCATCAGTACTGTTATTGATAACTTCTCTTGTCTTGCTGGCTCTCTCCATCAGCCAAATGGTGTATTATCGTCGGCACTGGCACGGTGACGTCGAGCAGCAGGCTCTCGACAAGCGCTCAGTGGAGGACCAGCCCAGTTCTATTGCTTCTGCCTGAAGAAGTCGCCTTGAAGGAGCCTTCAG // NRC-112 Witch Flounder GC1.4 (SEQ ID NO: 110)GCCCACTTTGTATTCGCAAGGTAAGAGCAATATATTTCAAATTCATTTAGACGAGACCAAGCATTTGGGATCTGTGCTCAACTTGTAACTGTATAATGCAAATGTTAACAATATTCTTCTTCTGTTGTTTTTGTAGAATGAAGTTCGCTGCCGCCTTCCTCATGATGTTCATGGTCGTCCTCATGGCTGAACCCGGAGAGGGTGCTTGGATGCCTGCCTTGAATAGGATCTATCATGGTAGAGTCACAGAGTTAATTTGCTTTTTACATTGCAAATATTTTAATATAACATGGCTGGAAAATCACAAAAATGAGTACTCGATATATTTGGCAAAGTAGAATCCCTTTGATTTCAATAATAATCAAAAACACAATCAAAAAGGCCATTGATTAGCATGTTCCTTCAATGAAATGGACATTGTAGTTTATTFTGATTCTGACATGCACCAACTTGCTGCGGCAACAATTGAATTCAAATTTGTCTCAGAAAAATTTAAAGTACATTTTTCTTTCCATTAATCGGATTTGTTTTAAAAAATACAGAATAACTGGATCTTTATGCTAAAATAATAAATCATACATTCTGATTTTACCAGTCAAGATTGAACGCTACTTAAAAGTATGTATAAAACATCATCTGTATTGATAATTGTTTAACTTTTAACTAATAGTCCTAATAATTGTGTTATGGAAATGTATTCATTGTCATTTAATATCATTTGCTTGAATTTATCACCATGTGTTTTTGTTTGTTTTTACACAGCTCTACTGAGGATCAATCGGTAAGGACTTCTACCATCATTACTGTGTAATTTTAATAGTATTATCATCAGTACTGTTATTGATAACTTCTCTTGTCTTGCTGACTCTCTCCATCAGCCAAATGGTGTATTATCGTAGGCACTGGCACGGTGACGTCGAGCAGCAGGCTCTCGACAAGCGCTCAGTGGAGGACCAGCCCAGTTCTATTGCTTCTGCCTGAAGAAGTCGCCTTGAAGGAGCCTTCAG // NRC-113 Witch Flounder GcSc4B35 (SEQ ID NO: 111)ATGAAGTTCACTGCCACCTTCCTGGTGTTGTTCATGGTCGTCCTCATGGCTGGATCCGGAGAGTGTGGTTGGAAAAAGTGGTTCACTAAAGGTGCCAAGCACCTTGGCCAGGCGGCCATTAACGGTTTGGCCTCTTGCGAAGAGCAGCAAGAGCTCGACAAGCGCTCAGAGGATGACGAGCCCAGTGCTATTGTTTTTGAA // NRC-114 WitchFlounder GC3.6 (SEQ ID NO: 112)ATGAAGTTCACTGCCACCTTCCTGGTGTTGTTCATGGTCGTCCTCATGGCTGGATCCGGAGAGTGTGGTTGGAAAAAGTGGCTCCGTAAAGGTAGAGTCATGGATTTAATTTGCTTTTTACATTGCAAATACTTTAATATAACATAGTTGGAAAATCACAAAAATAAGTAGTCGATATATTTGGCCATATAGAATCACTTTGATTTCAATAATAATCAAAACAACAATCAAAAAGCCCATTGATTAGCATGTTCCTTCACTAAAATGGACATTGTCATTTATTTTGATTCTCACAGGCACCAACCTGCTGCGGCAACAATTGAAATCAAATTTGTCTCAGAAGAATTCAAAGTACATTGTTCTAGGCGATTTAATCTTTCCATTCATCGGATTTGTTTTTAAAAATATAGAATAACTGGATCTCTATGTTAAAATAATAAAACACACATTCTGATTTTACCTGTCAAGATTGAACACGACTTAAAAGTATGTATAAAACATCATCTGTATGTATAATTGTTTAACTGTCAACTAATAGTCCAAATAATTGTGTTATGGAAATGTATTCATTGTCATATAATATCATTTGCTTGAATTTATCACCATGTGTTTTTGTTTGTTTTTACACAGGTGCCAAGCACCTTGGCCAGGCGGCCATTAAGTAAGGACTTCTACCATCATTACTGTGTAATTTTAACAGTATTATCATCAGTACTGTTATTGACAACTACTCTTGTCTCTGTGACTCTCTCCAGGGGTTTGGCCTCTTGCGAAGAGCAGCAGGAGCTCGACAAGCGCTCAATGGATGACGAGCCCAGTGCTATTGTTTTTGACTGAAGAAGTCGCCTTGAAGAGCCTTCAG // NRC-115 Witch Flounder GC2.2 (SEQ ID NO:113)GCCCACTTTGTATTCGCAAGGTAAGAGCGATATATTTCAAACTCATATAGACGAGACCAAGCATTTGGGAAATGTGCTCAGCTTGTTACTGTATAATGCAAATGTTAACAATGTTTTTGTTCTGTTGTTTTTGCAGAATGAAGCTCGCTGCTGCCTTCCTGGTGTTGTTCATGGTCGTCCTCATGGCTGAACATGGAGAGGGTTTTGGGGATTTCTATATGAAGCCTGGTAGAGTCACGGAATTAATTCGATTTTAACATGGCAAATATTTTACTATAACATACCATATGAGTAGTCGATTAATTAATTGGATTTGTTTTTAAAAATATAGAATAATTGGATCTTTATGCTAAAATAATTAAACATACATTCTGATTTTACCAGTTAAGATTGAACGCTACTTAAAAGTATGTATAAAACATCATCTGTACATATAATTGTTTAACTGTTAACCAATAGTCCAAATAATTGTGTTGTGGAAATGTATTAATTGTCATTTAATATCATTTGCTTGAATTTGTCACCATGTGTTGTTGTTTGTTTTTACACAGGTAGAAAGATTTCCCATGGGTAAGGACTTCTACCATCATTACTGTGTATTTTTAGCAGTATTATCATCAGTACTGTTATTGATAACTTCTCTTGTCTCGCTGACTCTCTACAGGTACATCAGAAGTCCTTATGGTTACGACGAGCAGCAGGAGGTCGACAAGCGCTCAGTCGATGACAACCCCAGTGCCATTGCTTCTGACTGAAGAAGTCGCCTTGAAGGAGCCTTCAGA // NRC-116 Witch Flounder GcSc4B28 (SEQ ID NO:114)ATGAAGTTCACTGCCACCTTCCTGGTGTTGTTCATGGTCGTCCTCATGGCTGAACCTGGCGAGGGTTATTGGCGCTTCCGCAACCACCGTGGTGAAAGGTTATCCCAGAGGCATTTCGCTGACGTCGAGCAGCAGGAGCTCGACAAGCGCTCAGTGGATGACGAGCCCAGTTCTATTGCTTTTGA // NRC-117 Witch Flounder GC3.7 (SEQID NO: 115)ATGAAGTTCACTGCCACCTTCCTCGTGTTGTTCATCGTCATGTTTGAACCTGGAGAGTGTTTTTGGAATGCTTTTTCACCGGGTCCACCATGGTCGGGTCACGGAAGTAGTTCGATTTTTACATGGCAAATATTTAAATGAAACATACCATATGAGTAGTCGATATATTTGGCCAAGTAGAATCACTTTGACTTCAATAATAATCAAAAACATAATCAAAAAGCCCATTGATTAGCATGTTCCTTCAATGAAATGGACATTGAGGTTTATTTTGATTCTCACAGGCACCAACCTGCTGCGGCAACAATTGCATTCAAATTTGTCCCAAAGAAACTTAATTAACATTTTCTGGCGATTTAATCTTTGCATAAATTGGATTTGTTTTTAAAAATATAGAATAACTGGATCTTTATGCTCAAATAATTAATCATACATTCTTATTTTATCAGTCAAGATTGAACGCTACTTAAAAGTATGTATAAAACATCATCTGTATGTATAATTGTTTAACTTTTAACTAAAAGTCCTAATAATTGTGTTATGGAAATGTATTAATTGTCATTTAATATCATTTCCTTGAATTTATCACCATGTGTTTTTGTTTGGTTTTTACACAGCTGGAAGGTTGATCCATAGGTAAGGACTTCTACCATCATTACTGTATAATGTTAATAATAGCATTATCATCAGTACTGTTATTGATAACTTCTCTTGTCTCGCTGACTCTCTCCATCAGATTCATCAAACGTCACGGTGACGTCGAGCAGCAGGAGCTCGACAAGCGCTCAGTGGATGACGAGCCCAGTTCTATTGCTTTTGCCTGAAGAAGTCGCCTTG // NRC-118 Witch Flounder GC3.1 (SEQ ID NO: 116)ATGAAGTTCACTGCCACCTTCCTGGTGTTGTTCATGGTCGTCCTCATGGCTGAACCTGGAGACTGTATTTTTGGATTGATTGCGACTGCGGTCCACAATGGTAAGTCAAGGAATTAATTCGATTTTTACGTGGCAAATATTTTAGTATAACATACCTTATGAGTAGTCGATATATTTGACCAAGTAGAATCATTTTGACTTCAATAATAATCAAAATAACAATCTCTAGGCAATTTAATATTTGCATTAATTGGATTTGTTTTTAAAAATATAGAATAACTGGATCTTAATGCTAAAATAATTAAACATACATTCTGATATTACCAGTCAAGATTGAACGCTACTTAAAAGTATGTATAAAACATCATCTGTATGTATAATTGTTTAACTGTCGACTAATAGTCCTAATAATTGTGTTATGGAAATGTATTCATTGTCATATAATATCATTTGCTTGAATTTATCACCATGTGTTTTTGTTTGTTTTTACACAGCTGGAAGGTTGATCCATAGGTAAGGACTTCTACCATCATTACTGTATAATTTTAAGAGCATTATCATCAGTACTGTTATTGATAACTTCTGTTGTCTCGCTGACTCTCTCCATCAGACTACTCGGCTTTCATCATGGGCCTCCCGGGTTCTGGCACGGTGACGTCGAGCAGCAGGAGCTCGACAAGCGCTCAGTGGATGAGGAGCCCAGTTCTATTGCTTTTGACTGAAGAAGTCGCCTTGAAGGAGCCTTCAG //NRC-119 Witch Flounder GC4.1 (SEQ ID NO: 117)ATGAAGTTCACTGCCACCTTCCTGGTGTTGTTCATGGTCGTCCTCATGGCTGAACCTGGAGACTGTATTTTTGGATTGATTGCGACTGCGGTCCACAATGGTAAGTCAAGGAATTAATTCGATTTTTACTTGGCAAATATTTTAGTATAACATACCTTATGAGTAGTCGATATATTTGACCAAGCAGAATCATTTTGATTTCAATAATAATCAAAATAACAATCTCTAGGCAATTTAATATTTGCATTAATTGGATTTGTTTTTAAAAATATAGAATAACTGGATCTTAATGCTAAAATAATTAAACATACATTCTGATATTACCAGTCAAGATTGAACGCTACTTAAAAGTATGTATAAAACATCATCTGTATGTATAATTGTTTAACTGTCGACTAATAGTCCTAATAATTGTGTTATGGAAATGTATTCATTGTCATATAATATCATTTGCTTGAATTTATCACCATGTGTTTTTGTTTGTTTTTACACAGTTGGAAGGTTGGTCCATGGGTAAGGACTTCTACCATCATTACTGTATAATTTTAAGAGCATTATCATCAGTACTGTTATTGATAACTTCTCTTGTCTCGCTGACTCTCTCCATCAGACTACTCGGCTTTCATCATGGGCCTCCCGGGTTCTGGCACGGTGACGTCGTGCAGCAGGAGCTCGACAAGCGCTCAGTGGATGAGGAGCCCAGTGCTATTGTTTTTGAATGAAGAAGTCGCCTTGAAGGAGCCTTCAG //NRC-120 Witch Flounder GC4.4 (SEQ ID NO: 118)ATGAAGTTCACTGCCACCTTCCTGGTGTTGTTCATGGTCGTCCTCATGGCTGAACCTGGAGACTGTATTTTTGGATTGATTGCGACTGCGGTCCACAATGGTAAGTCAAGGAATTAATTCGATTTTTACGTGGCAAATATTTTAGTATAACATACCTTATGAGTAGTCGATATATTTGACCAAGTAGAATCATTTTGGTTTCAATAATAATCAAAATAACAATCTCTAGGCAATTTAATATTTGCATTAATTGGATTTGTTTTTAAAAATATAGAATAACTGGATCTTAATGCTAAAATAATTAAACATACATTCTGATATTACCAGTCAAGATTGAACGCTACTTAAAAGTATGTATAAAACATCATCTGTATGTATAATTGTTTAACTGTCGACTAATAGTCCTAATAATTGTGTTATGGAAATGTATTCATTGTCATATAATATCATTTGCTTGAATTTATCACCATGTGTTTTTGTTTGTTTTTACACAGTTGGAAGGTTGGTCCATGGGTAAGGACTTCTACCATCATTACTGTATAATTTTAAGAGCATTATCATCAGTACTGTTATTGATAACTTCTCTTGTCTCGCTGACTCTCTCCATCAGACTACTCGGCTTTCATCATGGGCCTCCCAGGTTCTGGCACGGTGACGTCGAGCAGCAGGAGCTCGACAAGCGCTCAGTGGATGAGGAGCCCAGTGCTATTGTTTTTGAATGAAGAAGTCGCCTTGAAGGAGCCTTCAG //NRC-121 Petrale sole 02A (3) (SEQ ID NO: 119)ATGAAGTTCACTGCCACCTTCCTCGTGTTGTTCATGGTCATCGTCATGTTTGAACCTGGAGAGTGTTTTTTTGGAATGCGTTTTCACGGGGTCCACCATGGTAGGGTCACAAAAGTGATTTGATTATTACATGCCAAATATGTTAATGAAACATACCATATGAGCAGTCGTATTATTTGGACAAGTAGAATCACTTTGATTTCAATAGTAATTAAAATAACAATCAAAAAGGCCTTTGATTAGCATGTTCCTTCAATGAAATGGACATTGAGGTTTATTTTGATTCTCACCTGCATCGACCTGCTGCGGCAACTATTGAAATCAAATTTGTCCCAGAAGAAACTAAATTAACATTTTCTAGGCCATCTAATCTTTGCATGAATTGGATTTGCTTTCAAAAATATAGAATAACTGGATATTTATGCTAAAATAATAAAAACACACATTCTGATTTTACCAGTCAAGATTGAACACTACTTAAAAGTACGTATAAAACATCATCTGTATGTATAATTGTTTGACTTTTAACAAATAGTCAAAATGATTGTTATGGAAATGCATTAATTGTCATTTAATATCATTTACTTGAATTTATCACCATGTGTTTGTTTGTTTTTTAGCAGGTGGAGGTTTTCTCAATGCGCAAGGACTTCTACCATCATTACTGTGTAATTTTAATAGTATTATCATCAGTACTCTTATTGACAACGTCTCTTGTCTCGCTGACTCTCTCTATCAGATTAAACCCAGGGTATCGCGGTTACGACGAGCAGCAGGAGCTCGACAAGCGCGCAGTCGATGA // NRC-122 Petrale sole 02B(SEQ ID NO: 120)ATGAAGTTCACTGCCACCTTCCTGGTGTTGTCCTTGGTCGTCCTCATGGCTGAACCTGGAGAGGGTTTCTTTGGAGCCCTTCTCAAAGGTAGAGTCACGGAATTAATTTGATTGTTACATGGCAAATAATTTTGTATAACATATCATATGAGCAGTCGATGTATTTGACCAAGAAGAATCATTTTGATTTCAATAATAATCAAAATAACAATCTCTTGGAGATTATATATTTGCAATAATTGGATTTTATAAAATATAGAACAACTGGATCTTAATGCTAAAATAATTTAACATACATTCTGATTTTACCAGTCAAAATTAACCACTACTTTAAAGTATGTATAAAACATCATCTGTATGTTTAATTGTTTAACTTTTAACAAATAGTCCAAATAATTGTGTAATGGAAATGTATTCATTGTCATATAATATAGTTTGCTTGACTTTATCACCGTGTGTTTTTGTTTGTTTTTTCACAGGTGCCCAGGCGCTCCATGGGTAAGGACTTCTACCATCATGACTGTGTAAGTTTAATAATATTATCATCAGTACTGTTATTAACGACTTCTCTTGTCTCGCTGACTCTCTCCATCAGAATCATCCACAATGCTCGTCACGGTTACGACGAGCAGCAGGAACTCAACAAGCGCGCAGTCGATGA // NRC-123 Petralesole PL1/2/2.1 (SEQ ID NO: 121)GCCCACTTTGTATTCGCAAGGTAAGATCAATATTTTTCAAATTCATTTAGACGAGACCAACCGTTTGCGAAATGTGCTCAGCTTGTTATTGTATAATAACAAAGTTAACGATCTTTATTTTTCTGTTTTTTTGTAGAATGAAGTTCACTGCCACCTTCCTGATGTTGTTCATCTTCGTCCTCATGGTTGAACCTGGAGAGTGTGGTTGGAAAGATTGGTTTCGTAAGGCTAAGAAAGGTAGAATCACGGAATTAATTAGCTTTTTACATTGCAAATAGATTTTTTATAACAGCTGGAAATCACAAAAATAAATAGTCGATATATTTGGCCAATTAGAATCACTTTAATTTCAATAATAATCTAAATAACAACCTAAAAGGCCTTTGATTAGCATGTTCCTTCAATGAAAAGGACATTGAGGTTTATTTTGATTCTCACATGCACCGACCTGTGCGGCAACAATTGAATTCAGATTTGTCCCAGAAGAATTCAAAGTACATTTTTCCAGGCGATTAAATCTTTCCATTACTCGGATTTAAAAATAAATAAATAGAATAACTGAAGCGCTATGATAAAATAATTACACATTCATTCTGATTTTACAAGTCAAGATTGAACACTATTAAAAAGTGTGTATAAAACATCATCTGTATGTATAATTGTTTAACTGTTAATAGTCTTAATAATTGTGTTATGGAAATGTATTAATTTACATTTAATATCATTTGCTTGAGTTTACCATCATGTGTTTTTGTTTGTTTTTACACAGTTGGCAAGACTGTTGGCGGCTTGGCCCTTAAGTAAGAACTTCTACCATCATTACTGTATAATTTTGATAGTATTATCACCAGTACTGTTATTAACTACTTCTCTTGTCTCGCTGACTCTCTCCATCCGACTCATCCGCAGTCATTACCTTGGCGAGCAGCAGGAGCTTGCCAAGCGCGCAGTCGATGACGACCCCAGTGTTATTGTCTTTGACTGAAGAAGTCGCCTTGAAGGAGCCTTCAG // NRC-124 English sole 05A (SEQ ID NO: 122)ATGAAGTTCACTGCCACCTTCCTCATGATTTTAATCTTCGTCCTCATGGTCGAACCTGGAGAGTGTGGTATTAGGAAATGGTTTAAAAAGGCTGCTCACGGTAAAGTCACGGAATTAATTTGCTTTTTGCTTTACAAATATTTTTTTATAGCAGCTGGAAAATCACAAAAATAAATAGTCGATGTATTTGGCCAATTAGAATCACTTTGATTTCAAATAATAATCTAAATAGCAACCTAAAAGGCCTTTGATTAGCATGTTCCTTCAATGAAATGGATGTTGAGGTTTATTTTGATTCTCACATGCACCGACCTGCTGCGGCAACAATTGAATTCAAATTTGTCCCAAAGGAATTCAAAGTAAACTTTTCTAGATGATTTAATCTTTCCATAACTCGGCTTTGTTTTTAAAAATATATAATAACTCAATCACTATGATAAAATAATAACACATACATTCTGATTTATACAAGACAAGATTGAAAACTTCTTAAAAGTATGTATAAAACATCATCTGTTTGTATAATTGTTTATCATTTCACAAAAAGTCCAACTAATTGTGTTATGGAATTGTATAAATTGTCATTTAATATAATTTTTTTGAGTTTATCAATATGTGTTTTTGTTTGTTTTACACAGTTGGCAAGGAAGTTGGCAAGGTGGCCCTTAAGTAAGGACTTCTACCATTATTACTGTATAATTTTGATAGTATTATCACCCGTACTGTTATTGACAACTTCTCTTTTCCTGCTGACTCTCTCCATCTGACTCATCTGCAGTGCTTGCCTTGACAAGCAGCAGCAGCTCGACAAGCGCGCAGTCGATGA //NRC-125 English sole PL1/2/5 (SEQ ID NO: 123)GCCCACTTTGTATTCGCAAGGTAATATCGATATTTTTCAAACTCATTTAGACGAGACCAAGCATTTGGGAAATGTGCTAAGGTTGTTACTGTATAATGCAAAATTAATGATCTTTATTTTTCTGTTTTTTTTTGCAGAATGAAGTTCACTGCCACCTTCCTCATGATTTTAATCTTCGTCCTCATGGTCGAACCTGGAGAGTGTGGTTTGAAGAAATGGTTTAAAAAGGCTGTTCACGGTAGAGTCACGGAATTAATTTGCTTTTTGCTTTACAAATATTTTTTTATAGCAGCTGGAAAATCACAAAAATAAATAGTCGATGTATTTGGCCAATTAGAATCACTTTGATTTCAATAATAATCTAAATAGCAACCTAAAAGGCCTTTGATTAGCATGTTCCTTCAATGAAATGGATGTTGAGGTTTATTTTGATTCTCACATGCACCGACCTGCTGCGGCAACAATTGAATTCCAATTTGTCCCAAAGGAATTCAAAGTAAACTTTTCTAGGCGATTTAATCTTTCCATAACTCGGCTTTGTTTTTAAAAATATATAATAACTCAATCCCTATGATAAAATAATAACACATACATTCTGATTTATACAAGACAAGATTGAAAACTTCTTGAAAGTATGTATCAAACATCATCTGTTTGTATAATTGTTTAACAGTTCACAAAAAGTCCAACTAATTGTGTTATGGAATTGTATAAATTGTCATTTAATATAATTTTTTTGAGTTTATCAATATGTGTTTTTGTTTGTTTTACACAGTTGGCAAGAAAGTTGGCAAGGTGGCCCTTAAGTAAGGACTTCTACCATTATTACTGTGTAATTTTGATAGTATTATCACCAGTACTGTTATTGACAACTTCTCTTTTCCTGCTGACTCTCTCCATCCGACTCATCTGCAGTGCTTACCTTGGCGAGCAGCAGCAGCTCGACAAGCGTGCAGTCGATGAAGAGCCCAGTGTTATTGCTTTTGACTGAAGAAGTCGCCTTGAAGGAGCCTTCAG // NRC-126 Starry flounder 09A (SEQ ID NO:124)ATGAAGTTCACTGCCACCTTCCTCATGATGTTCATCTTCGTCCTCATGGTTGAACCTGGAGAGTGTGGTTGGAGGAAATGGATTAAAAAGGCTACTCACGGTAAAGTCACGGAATTAATTCGTTTTTTGCTTTGCAAATATTTTTTTTATAACAGCTGGAAAGTCACAAAAATAAATAGTCAATATATTTGGCCAATTAGAATCACTTTGAGTTCAATAATAATCTAAATAACAACCAAAAAGGCCTTTCCTTTAATGAAATGTACGTTGAAGTTTATTTTGAATCTCACATGCACCGACCTGCTGCGGCAACAATTGAATTCAAATTTCTCCCAGAGGAATTCAAAGTAAATTTTTCTAGGCGATTTAATCTTTCCATTACTCTGATTTGTTTTAAATATATAGAATGACTCAATTGCTATGATAAAATAATAAGCCATACATTCTGATTTTTACAAGACAAGATTGAAAACTTCTTAAAAGTACGTATAAAACATCATCTGTATTTATAATTGTTTAACATTTAACAAATTGTCCTACTAATTGTGTTATGGAAATGTATAAATTGTCATTTAATATCATTTGCTTGAGTTTATCATTATTTGTTTTTGTTTGTTTTTACACAGTTGGCAAGCATATTGGCAAGGCGGCCCTTGAGTAAGAACTTCTACCATCATTACTGTATAATTTTGATAGTATTATCACCAGTACTGTTATTGACAACTTCTCTTGTCCTGATGACTCTGTTCATCCAACTCATCTGCAGTGCTTACATTGGCGGGAAGCAAGAACTCGACAAGCGCGCAGTCGATGA // NRC-127 (SEQID NO: 327)ATGAAGTTCACTGCCACCTTCCTCATGATTTTAATCTTCGTCCTCATGGTCGAACCTGGAGAGTGTGGTTGTAAGAAATGGTTTAAAAAGGCTGCTCACGGTAGAGTCACGGAATTAATTTGCTTTTTGCTTTACAAATATTTTTTTATAGCAGCTGGAAAATCACAAAAATAAATAGTCGATGTATTTGGCCAATTAGAATCACTTTCATTTCAATAATAATCTAAATAGCAACCTAAAAGGCCTTTGATTAGCATGTTCCTTCAATGAAATGGATGTTGAGGTTTATTTTGATTCTCACATGCACCGACCTGCTGCGGCAACAATTGAATTCCAATTTGTCCCAAAGGAATTCAAAGTAAACTTTTCTAGGCGATTTAATCTTTCCATAACTCGGCTTTGTTTTTAAAAATATATAATAACTCAATCCCTATGATAAAATAATAACACATACATTCTGATTTATACAAGACAAGATTGAAAACTTCTTGAAAGTATGTATCAAACATCATCTGTTTGTATAATTGTTTAACATTTCACAAAAAGTCCAACTAATTGTGTTATGGAATTGTATAAATTGTCATTTAATATAATTTTTTTGAGTTTATCAATATGTGTTTTTGTTTGTTTTACACAGTTGGCAAGAACGTTGGCAAGGTGGCCCTTAAGTAAGGACTTCTACCATTATTACTGTATAATTTTGATAGTATTATCACCAGTACTGTTATTGACAACTTCTCTTTTCCTGCTGACTCTCTCCATCCGACTCATCTGCAGTGCTTACCTTGGTGAGCAGCAGCAGCTCGACAAGCGTGCAGTCGATGAAGAGCCCAGTGTTATTGCTTTTGACTGAAGAAGTCGCCTTGAAGGAGCCTTCAG // NRC-128 (SEQ ID NO: 129)GCCCACTTTGTATTCGCAAGGTAATATCGATATTTTTCAAACTCATTTAGACGAGACCAAGCATTTGGGAAACGTGCTAAGGTTGTTACTGTATAATGCAAAATTAATGATCTTTATTTTTCTGTTTTTTTTTGCAGAATGAAGTTCACTGCCACCTTCCTCATGATTTTAATCTTCGTCCTCATGGTCGAACCTGGAGAGTGTGGTATTAGGAAATGGTTTAAAAAGGCTGCTCACGGTAAAGTCACGGAATTAATTTGCTTTTTGCTTTACAAAATATTTTTTTATAGCAGCTGGAAAATCACAAAAATAAATAGTCGATGTATTTGGCCAATTAGAATCACTTTGATTTCAATAATAATCTAAATAGCAACCTAAAAGGCCTTTGATTAGCATGTTCCTTCAATGAAATGGATGTTGAGGTTTATTTTGATTCTCACATGCACCGACCTGCTGCGGCAACAATTGAATTCAAATTTGTCCCAAAGGAATTCAAAGTAAACTTTTCTAGGCGATTTAATCTTTCCATAACTCGGGCTTTGTTTTTAAAAATATATAATAACTCAATCCCTATGATAAAATAATAACACATACATTCTGATTTATACAAGACAAGATTGAAAACTTCTTGAAAGTATGTATCAAACATCATCTGTTTGTATAATTGTTTAACATTTCACAAAAAGTCCAACTAGTTGTGTTATGGAATTGTATAAATTGTCATTTAATATAATTTTTTTGAGTTTATCAATATGTGTTTTTGTTTGTTTTACACAGTTGGCAAGAAAGTTGGCAAGGTGGCCCTTAAGTAAGGACTTCTACCATTATTACTGTATAATTTTGATAGTATTATCACCAGTACTGTTATTGACAACTTCTCTTTTCCTGCTGACTCTCTCCATCCGACTCATCTGCAGTGCTTACCTTGGCGAGCAGCAGCAGCTCGACAAGCGTGCAGTCGATGAAGAGCCCAGTGTTATTGCTTTTGACTGAAGAAGTCGCCTTGAAGGAGCCTTCAG // NRC-129 (SEQ ID NO: 130)AATGAAGTTCACTGCCACCTTCCTCATAGAATGGTTCATCTTCGTCCTCAATGGGTTGAAACCTGAAGAAGTGTGGTTGGAAAGAAAGTGGTTTAAAAAGGCTACTCACGGTAAAGTCACGGAATTAATTAGCATTTTTCTTTGCAAATATTTTTTTTATACAGCTCGAAAATTCACAAAAATAAATAGTCGATATATTTGGCCAATTAGAATCACTTTGATTTCAATAATAATCTAAATAACAACCTAAAAGGCCTTTGATTAGCATGTTCCTTCAATGAAATGGACGTTGAGGTTTATATTGATTCTCACATGCACCGACCTGCTGCGTCAACAATTGAATTCAAATTTGAGAGGAATTCAGCGTAAATTTTTCTAGGCGATTTAATCTTTCCATTACTCGGATTTGTTTTTAAATATATAGAATAACTCAATTGCTATGATAAAATAATAACACATACATTCAGATTTTTACAAGACAAGATTGAAAACTTCTTAAAGGTACGTATAAAACATCATCTGTATTTATAATTGTTTAACATTTAACAAATAATCCTACTAATTGTGTTATGGAAATGTATAAATTGTAATTTAATATAATTTGCTTTAGTTTATCATTATTTGTTTTTGTTTGTTTTTACACAGTTGGCAAGCATGTTGGCAAGGCGGCCCTTGAGTAAGAACTTCTACCATCATTACTGTATAATTTTGATAGTGTTATCACCAGTACTGTTATTGACAACTTCTCTTGTCCTGCTGACTCTCTCCATCCGACTCATCCGCAGTGCTTACCTCGGCGAGAAGCAAGAACTCGACAAGCGCGCAGTCGATG// NRC-130 Greenland halibut 12B (SEQ ID NO: 131)ATGAAGTTCACTGCCACCTTCCTGGTGTTGTTCATGGTCGTCCTCATGGCTGAACCTGGAGAGGGTTTTTTCGGATTGCTTTTTCACGGGATCCACCATGGTAGGGTCACGGAATTAATTAGATGTTTACATGGCAAATATTTTAAGATAACACACCATATGAGTAGTCGATATATTTGACCAATTAGAATCACTTTAATTTCAATAATAATCACAATAACAATCTCTAGGCCATTTAATCTTTCCATTAATCGGATTTGTTTTTTTAAATATAGAATAACTGGATCTTTATGCTAAAATAATGAAACATACATTCTGATTTTACCAGTCAAGATTGAACGTTACTTAAAAGTATGTTTAAAACATCATCTGTATGTATAATTGTTTAGCTGTAAACAAATAGTCCAAATAATTGTGTTATGGAAATGTATTAATTGTCATATAATATAATTTGCTTGAATTTATCACCATGTGTTTTTGTTTGTTTTTTAACACAGCTGGAAAGTTGATCCATGGGTAAGGACTTCTACCATCATTACTGTGTATTTTTAATAGTATTATCATCAGTACTGTTATTAACAACTTCTCTTCTATCGCTGACTCTCTCCATCAGACTCATCCATCATGGTTACGACGAGCAGCAGGAGCTCGACAAGCGCGCAGTCGATGA //NRC-131 Pacific Halibut 15A (SEQ ID NO: 132)ATGAAGTTCACTGCCACCTTCCTGGTGTTGTTCATGGTCGTCCTCATGGCTGAACCTGGAGAGGGTTTGGGAAATTGGATGGGGCCCCATATCAGCGGTAGAGTCACGGAATTAATTTGCTTTTTCCATTGCAAATATTTTAATATTGCATAGCTGGAAAATCACGAAATAAGTAGTCGATATATTTGGCCAAATAGAATAACTTTGATTTCAATAATAATCAAAATTACAATCAAAAAGGCCTTTGATTAGCATGTTCCTTCAATAAAATGGACATTGAAGTTTATTTTGATGCTCACATGCACCGACCTGCTGCGGCAACAATTGAAATCAAATTTGTCTCAGAATTTAAAGTACATTTTTCTAGGTGATTTAATCTTTCCATTCATCTGATTTATTTTATAAATATAGAATAACTGGATCTTTCTGCTAAAATAATAAAACACACATTCTGATTTTACCAGTCAAGATTGAACACTACTTAAAAGTATGTATAAAACATCATCTGTATGTATAATTGTTTAACTGTTAACAATAGTCCAAATAATTGTGTTAAGGAAATGTATTAATTGTCATTTAATATCATTTGCTTGAATTTATCACCATGAGTTTTTTGTTTGTTTTTACACAGGTAGAAAGAAGGCCTTGCAGTAAGGACTTCTACCATCATTACTTTGTAATTTTTATAGTATTATCATCAGTACTGTTATTGACAACTTCTCTTGTCTCGCTGACTCTCTCCATCAGGATGAACTCAGAGCGTCGCAGTTACGACGAGTAGCAGCAGAAGCTCGACAAGCGCGCAGTCGATGA // NRC-132 PacificHalibut 15B (SEQ ID NO: 133)ATGAAGTTCACTGCCACCTTCCTGGTGTTGTTCATGGTCGTCCTCATGGCTGAACCTGGAGAGTGTTTTTTGGGATTGCTTTTTCACGGGGTCCACCATGGTAGGGTCACGGAAGTAATTCGATTTTTACATGGCAAATATTTTAAGATAACACACCATATGAGTAGTCGATATATTTGATATATTAGAATCACTTTGATTTCAATAATAATCAAAATAACAATCTCTAGGCGATTTAATATTTGCATTAATTGGATTTGTTTTTAAAAATATAGAATAACTGGATCTTTATGGTAAAATAATTAAACATACATTCTGATTTTACCAGTCAAGATTGAACACTACTTAGAAGTATGTATAAAACATCATCTGTATGTATAATTGTTTAACTGTTAACTAATAGTCCAAATAATTGTGTTATGGAAATGTATTAATTGTCATTTAATATCATTTGCTTGAATTTATCACCATGTGTTTTTGTTTGTTTTTACACAGTTGGAAATTTGATCCATGGGTAAGGACTTCTACCATCATTACTGTGTATTTTTAATAGTATTATCATCAGTACTGTTATTGACAACTTCTCTTGTCTCGCTGACTCTCTCCATCAGACTCATCCATCACGGTTACGACGAGCAGCAGGAGCTCGACAAGCGCGCAGTCGATGA // NRC-133C 0 sole PL1/2/6 (SEQ ID NO: 134)GCCCACTTTGTATTCGCAAGGTAATATCGATATTTTTCAAACTCATTTAGACGAGACCAGGCATTTGGGAAACGTGCTAAGGTTGTTACTGTATAATGCAAAATTAATGATCTTTATTTTTCTGTTTTTTTTTGCAGAATGAAGTTCACTGCCACCTTCCTCATGATTTTAATCTTCGTCCTCATGGTCGAACCTGGAGAGTGTGGTATTAGGAAATGGTTTAAAAAGGCTGCTCACGGTAAAGTCACGGAATTAATTTGCTTTTTGCTTTACAAATATTTTTTTACAGCAGCTGGAAAATCACAAAAATAAATAGTCGATGTATTTGGCCAATTAGAATCACTTTGATTTCAATAATAATCTAAATAGCAACCTAAAAGGCCTTTGATTAGCATGTTCCTTCAATGAAATGGGTGTTGAGGTTTATTTTGATTCTCACATGCACCGACCTGCTGCGGCAACAATTGAATTCAAATTTGTCCCAAAGGAATTCAAAGTAAACTTTTCTAGGCGATTTAATCTTTCCATAACTCGGCTTTGTTTTTAAAAATATATAATAACTCAATCGCTATGATAAAATAATAACACATACATTCTGATTTATACAAGACAAGATTGAAAACTTCTTGAAAGTATGTATCAAACATCATCTGTTTATATAATTGTTTAACATTTCACAAAAAGTCCAACTAATTGTGTTATGGAATTGTATAAATTGTCATTTAATATAATTTTTTTGAGTTTATCAATATGTGTTTTTGTTTGTTTTACACAGTTGGCAAGAAAGTTGGCAAGGTGGCCCTTAAGTAAGGACTTCTACCATTATTACTGTATAATTTTGATAGTATTATCACCAGTACTGTTATTGACAACTTCTCTTTTCCTGCTGACTCTCTCCATCCGACTCATCTGCAGTGCTTACCTTGGCGAGCAGCAGCAGCTCGACAAGCGTGCAGTCGATGAAGAGCCCAGTGTTATTGCTTTTGACTGAAGGAGTCGCCTTGAAGGAGCCTTC //

TABLE 13 Nucleotide sequences encoding hepcidin-like peptides of Table11. NRC201 (SEQ ID NO: 135)CGCCCTTAAGATGAAGACATTCAGTGTTGCAGTTGCAGTGGTGGTCGTCCTCGCATGTATGTTCATCCTTGAAAGCACCGCTGTTCCTTTCTCCGAGGTGCGAACGGAGGAGGTTGAAAGCATTGACAGTCCAGTTGGGGAACATCAACAGCCGGGCGGCACGTCCATGAATCTGCCGGTACGTTCAATTTAGTGAATGAATTAAGTAATTACCTTTAGCAAATTAACATCTAAGTGGTTGCGTTTCACCCTTGGAATTGAATTAGCCCACTAGCGCTAGTTGTTAACCATTTGATTGTGAGCCGGTAGAGAGGGCTTCAGGGCGAGTAGTGTGAATACTTGTGAAGTGGAGACTTGGACAAAAATACTTACCATGTGCTTGTTCCCACCTTTTTCATTTTCTTTTCTTGGCTGAGATACAGATGCATTTCAGGTTCAAGCGTCAGAGCCACCTCTCCCTGTGCCGTTGGTGCTGCAACTGCTGTCACAACAAGGGCTGTGGCTTCTGCTGCAAATTCTGAGGACCTGCCAGCAAAGGGCGAATTCGTTTAAAACAC // NRC202(SEQ ID NO: 136)AGATGAAGACATTCAGTGTTGCAGTTGCAGTGGTGGTCGTCCTCGCATGTATGTTCATCCTTGAAAGCACCGCTGTTCCTTTCTCCGAGGTGCGAACGGAGGAGGTTGAAAGCATTGACAGTCCAGTTGGGGAACATCAACAGCCGGGCGGCACGTCCATGAATCTGCCGATGCATTTCAGGTTCAAGCGTCAGAGCCACCTCTCCCTGTGCCGTTGGTGCTGCAACTGCTGTCACAACAAGGGCTGTGGCTTCTGCTGCAAATTCTGAGGACCTGCCAGCA //NRC203 (SEQ ID NO: 137)ACGAGGTCCCTCATCCGCTGACACCAAAAGAACAATCAATCAACTTTGGACTCGTCTTAGTGCATTGAAAATTGTGCGTTGGAGAGCGTCGCTTTTTGGGAACATTGAAGAGTTCTGATCTTCCTCATAAACTGTCACTTCAATTTCAACTGATTTCAACAGGACTTTTAAATAGGCTATAAACTTCCTAAAAAAAACGAGAATGAAGGCCTTTAGTGTTGCAGTGGTACTCGTCATTGCATGTATGTTCATCCTTGAAAGCACCGCTGTTCCTTTCTCCGAGGTGCGAACGGAGGAGGTTGGAAGCTTTGACAGTCCAGTTGGGGAACATCAACAGCCGGGCGGCGAGTCCATGCATCTGCCGGAGCCTTTCAGGTTCAAGCGTCAGATCCACCTCTCCCTGTGCGGTTTGTGCTGCAACTGCTGTCACAACATTGGCTGTGGCTTCTGCTGCAAATTCTAAGGACCTGCCCGCAACATTTTCTAGTTTGTACATGTTTGCAATGTTTTCTTTCTGAGATGTTGTTTTTGTGACTATGATAATGATTTATAAAATCACTTCTTATTGTGACACTTTAAAAAAAATAAACACATTCTTTGAATACAAAAAAAAAAAAAAAAAA //NRC204 (SEQ ID NO: 138)CGAACGGAGGAGGTTGAAAGCATTGACAGTCCAGTTGGGGAACATCAACAGCCGGGCGGCACGTCCATGAATCTGCCGATGCATTTCAGGTTCAAACGTCAGAGCCACCTCTCCCTGTGCCGTTGGTGCTGCAACTGCTGTCACAACAAGGGCTGTGGCTTCTGCTGCAAATTCTGAGGACCTGCCAGCACTAAAGCCATTTTATTAACTTATCGCCTTTAATTTGCCCCTATTCTTCTATGTTTCTTTTGGACTCTGTGGAGAAGATGCAATCTCATTGACGTCTTTATCACTGCACAACCTCAATCTTGT // NRC205 (SEQ ID NO: 139)AAGATGAAGACATTCAGTGTTGCAGTGGTACCCGTCATTGCATGTATGTTCATCCTTGAAAGCACCGCTGTTCCTTTCTCCGAGGTGCGAACGGAGGAGGTTGGAAGCTTTGACAGTCCAGTTGGGGAACATCAACAGCCGGGCGGCACGTCCATGAATCTGCCGATGCATTTCAGGTTCAAGCGTCAGAGCCACCTCTCCCTGTGCCGTTGGTGCTTCAACTGCTGTCACAACAAAGGCTGTGGCTTCTGCTGCAAATTCTGAGGACCTGCCAGCA //NRC206 (SEQ ID NO: 140)TAAGATGAAGCAATTCAGTGTGGCAGTGGTACTCGTCATGGCATGTATGTTCATCGTGGAAAGCACCGCTGTTCCTTTCTCCGAGGTGCGAACGGAGGAGGTTGGAAGCTTGGACAGTCCAGTTGGGGAACATCAACAGCCGGGCGGCGAGTCCATGCATCTGCCGGAGCCTTTCAGGTTCAAGCGTCAGATCCACCTCTCCCTGTGCGGTTTGTGCTGCAACTGCTGTCACAACATTGGCTGTGGCTTCTGCTGCAAATTCTGAGACTGCCAGCA //NRC207 (SEQ ID NO: 141)ACGAGGCACACGCTGACCAGGGGGTCACCACAACTTCTGAAGAGACCCAGGTTCCTAGAGAGCCACTAGAGAATCACCCGGGAGCCCGAAGAACACAGGACGCTGCGGTGCTCGTCGGTGGCCGGACACCCATGAGACAGAAGACCTACAAGCCTCTCAGCTTCAGAAGGATTTCCTGACTCAGCATCTAAAACCTCCCTCAAAATGAAGGCATTCAGCATTGCAGTTGCAGTGACACTCGTGCTCGCCTTTGTTTGCATTCAGTGCAGCTCTGCCGTCCCATTCCAAGGGGTGCAGGAGCTGGAGGAGGCCGGGGGCAATGACACTCCAGTTGCGGAACATCAAGTGATGTCAATGGAATCCTGGATGGAGAATCCCACCAGGCAGAAGCGCCACATCAGCCACATCTCCCTGTGCCGCTGGTGCTGCAACTGCTGCAAGGCCAACAAGGGCTGTGGCTTCTGCTGCAAGTTCTGAGGATTCCCGCAACACAACCTCACAATGTATTAATTTATTACACTTTTTGTCGAGAAATGTCCTTTTTCTTGACCTCTTTTGTAATTTTGTATAATCTTTTAAATAAAACGGGGTACGATTCATGGAAAAAACCCTTTGAATAAAATAAAAAAAAAAAAAAAAAAAAAAAC // NRC208 (SEQ ID NO: 142)AAGATGAAGACATTCAGTGTTGCAGTTGCAGTGACACTCGTGCTCGCCTTTGTTTGCATTCAGGACAGCTCTGCCGTCCCATTCCAGGGGGTAAGAACGCAACTTTAACTCGCTTCATTTGCTTATTAGCCATAAATGTTTTGTCAGGATGCTGAGACACGGCTCCTAAATGTGTATAATTCATTAACAGGTGCAGGAGCTGGAGGAGGCAGGGGGCAATGACACTCCAGTTGCGGCACATCAAATGATGTCAATGGAATCGTGGATGGTATGTTCAATCTGTTCAATCGACTGGATGAATTAAGCCAATTACTGTGAGCGCGTTAACATTTAAGTGGCTGTGTTCCAGCCCGGTGGTGTAGGGAATAAAACCCCTCGTTCATGTGTCTTGTCCGTCCACAGGAGAGTCCCGTCAGGCAGAAGCGTCACATCAGCCACATCTCCATGTGCCGCTGGTGCTGCAACTGCTGCAAGGCCAAGGGCTGTGGCCCCTGCTGCAAATTCTGAGGACCTGCCCAGCA // NRC209 (SEQ ID NO: 143)AAGATGAAGACATTCAGTGTTGCAGTCACAGTGGCCGTCGTGCTCGTCTTTATTTGTATCCAGCAGAGCTCTGGCACCTTTCCTGAGGTAAGCTCCTGACTTCAGATCGTTTCATTTTGCTTGTTATCCATGAATCTCTCATCAACAGACTGAGACTTGATTCCTTCTTTATCAGGTACAAGAGCTGGAGGAGGCAGTGAGCAATGACAATGCAGCTGCTGAACATCAGGAGACATCAGTGGACTCATGGATGGTAGOTTCAGTTCACTGAATGGATCAAACCAATTCACATCAGACCTTTCAGATGGAAGTGAATGTGTTTTAGTCTCAAAGOTGCCCTGAAGCTCAGTTTACACAAGCAGTGAAAACAAACACAGAAAGTTATGATGATGCTGATGAACTTCTCCTCATGTCTCATGTCTCTCACACAGATGCCATACAACAGACAGAAGCGTGCCTTCAAGTGTAAGTTCTGCTGCGGCTGCTGCAGAGCTGGTGTCTGTGGACTGTGCTGCAAGTTCTGAGGATTCCTGCTCCAACAAC // NRC210 (SEQ ID NO:144)ACGAGCTGACAGGAGCTGACAGGAGTCACCAGCAGAGTCAAAGAACTAAACAACTTAACTCAGTCAAACTCTCAAAGATGAAGACATTCAGTGTTGCAGTCACAGTGGCCGTCGTCCTCGTCTTTATTTGTATCCAGCAGAGCTCTGCCTCCTTTCCTGAGGCACAAGAGCTGGAGGAGGCAGTGAGCAATGACAATGCAGCTGCTGAGCATCAGGAGACACCAGTGGACTCGTGGATGATGCCATACAACAGACAGAAGCGTAGCTTTAAGTGTAAGTTCTGCTGCGGCTGCTGCAGAGCTGGTGTCTGTGGACTGTGGTGCAAGTTCTGAGGATTCCTGCTCCAACAACCATCAAATATTCATTTGTTTTGCCTTTTGTCTTAAAGTTCATTGAACTATAAACATATTTCTGGTTGAGCATGTGATAGTTTAATGGTGTTACTCATTGGTTCATGGTATAGTCAAGTGTTCAGAGATGTGATTGTATCACCCACATATTTTCTCTGTTAGGTGTATTTTCAATAAATGCCAATGATCCTTTGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA // NRC211 (SEQ ID NO: 145)ACGAGCGGCACGAGGTGAACTGACAGGAGCTGACAGGAGTCACCAGCAGAGTCAAAGAACTAAACAACTTAACTCAGTCAAACTCTCAAAGATGAAGACATTCAGTGTTGCAGTCACAGTGGCCGTCGTGCTCGTCTTTATTTGTATCCAGCAGAGCTCTGCCTCCTTTCCTGAGGCACAAGAGCTGGAGGAGGCAGTGAGCAATGACAATGCAGCTGCTGAACATCAGGAGACACCAGTTGACTCGTGGATGATGCCAAACAACAGACAGAAGCGTGGCTTTAAGTGTAAGTTCTGCTGCGGCTGCTGCAGAGCTGGTGTCTGTGGACTGTGCTGCAAGTTCTGAGGATTCCTGCTCCAACAACCATCAAATATTCATTTGTTTTGCCTTTTGTTTTAAAGTTCATTGAACTATATACATATTTCTGGTAGAGCATGTGATAGTTTAATGGTGCTACTCCTTGGTTCATGGTGTAGTTAAGTGTTCAGAGATGTGATTGTATCACCCACATATTTCTCTGTTAAGGTGTATTTTCAATAAATGTTAATGCTCCTTTGAAAAAAAAAAAAAAAAAAAAAAA // NRC212 (SEQ ID NO: 146)ACGAGACTGACAGGAGCTGACAGGAGTCACCAGCAGAGTCAAAGAACTAAACAACTTAACTCAGTCAAACTCTCAAAGATGAAGACATTCAGTGTTGCAGTCACAGTGGCCGTCGTGCTCGTCTTTATTTGTATCCAGCAGAGCTCTGCCACCTTTCCTGAGATGCCATACAACAGACAGAAGCGTGGCTTTAAGTGTAAGTTCTGCTGCGGCTGCTGCGGAGCTGGTGTCTGTGGAATGTGCTGCAAGTTCTGAGGATTCCTGCTCCAACAACCATCAAATATTCATTTGTTTTGCCTTTTGTCTTAAAGTTCATTGAACTATAAACATATTTCTGGTTGAGCATGTGATAGTTTAATGGTGTTACTCATTGGTTCATGGTATAGTCAAGTGTTCAGAGATGTGATTGTATCACCCACATATTTTCTCTGTTAGGTGTATTTTCAATAAATGCCAATGATCCTTTGAAAAAAAAAA // NRC213 (SEQ IDNO: 147)AAGATGAAGACATTCAGTGTTGCAGTCACAGTGGCCGTCGTGCTCGTCTTTATTTGTATCCAGCAGAGCTCTGCCTCCTTTCCTGAGGTAAGCACCTGACTTCAGATCGTTTCATTTGCTTGTTATCCATGAATCTCTCATCATCATACTGAGACTTGATTCCTTCTTTATCAGGCACAAGAGCTGGAGGAGGCAGTGAGCAATGACAATGCAGCTGCTGAGCATCAGGAGACACCAGTGGACTCCAGGAGTGAATGTGTTTTAGTCAcAAAAGTGCCCTGAAGCTCAGTTTACACAAGCAGAGAAAACAAACAGAGTAAGTTATGATGATGCTGATGAAGGTCTCCTCATGTCTCATGTCTCTCACACAGATTCCATACAACAGACAGAAGCGTAGCTTTAAGTGTAAGTTCTGCTGCGGCTGCTGCAGAGCTGGTGTCTGTGGACTGTGCTGCAAGTTCTGAGGATTCCTGCTCCAACAAC // NRC214 (SEQID NO: 148)AGATGAAGACATGCAGTGTTGCAGTCACAGTGGCCGTCGTGCTCGTCTTTATTTGTATCCAGCAGAGCTCTGCCTCCTTTCCTGAGGTAAGCACCTGACTTCAGATCGTTTCATTTGCTTGTTATCCATGAATCTCTCATCATCATACTGAGACTTGATTCCTTCTTTATCAGGTACAAGAGCTGGAGGAGGCAGTGAGCAATGACAATGCAGCTGCTGAACATCAGGAGACACCAGTTGACTCGTGGATGGTAGGTTCAGTTCACTGAATGGATCAATCCATTTCACATCAGATCTTTCAGATGGAAGTGAATGTGTTTTAGTCACAAAAGTGCCCCTGAAGCTCAGTTTACACAAGCAGAGAAAACAAACAGAGTAAGTTATGATGATGCTGATGAAGGTCTCCTCATGTCTCATGTCTCTCACACAGATGCCAAACAACAGACAGAAGCGTGGCTTTAAGTGTAAGTTCTGCTGCGGCTGCTGCAGAGCTGGTGTCTGTGGACTGTGCTGCAAGTTCTGAGGATTCCTGCTCCGGACAA // NRC215 (SEQ ID NO: 149)AAGATGAAGACAATCAGTGTTGCAGTCACAGTGGCCGTCGTCCTCGTCTTTATTTGTATCCAGCAGAGCTCTGCCTCCTTTCCTGAGGTAAGCACCTGACTTCAGATCGTTTAATTTGCTTGTTATCCATGAATCTCTCATCAACATACTGAGACTTGATTCCTTCTTTATCAGGCACAAGAGCTGGAGGAGGCAGTGAGCAATGACAATGCAGCTGCTGAGCATCAGGAGACACCAGTGGACTCAGGGATGGTAGGTTCAGTTCACTGAATGGATCAATCCATTTCACATCAGATCTTTCAGATTGAAGTGAATGTGTTTTAGTCACAAAAGTGCCCTGAAGCTCAGTTTACACAAGCAGAGAAAACAAACAGAGTAAGTTATGATGATGCTGATGAAGGTCTCCTCATGTCTCATGTCTCTCACACAGATTCCATACAACAGACAGAAGCGTAGCTTTAAGTGTAAGTTCTGCTGCGGCTGCTGCAGAGCTGGTGTCTGTGGACTGTGCTGCAAATTCTGAGGACCTGCCAGCA // NRC216 (SEQ ID NO: 150)AAGATGAAGACATTCAGTGGTGCAGTCACAGTGGCCGTCGTGCTCGTCTTTATTTGTATCCAGCAGAGCTCTGCCTCCTTTCCTGAGGTAAGCACCTGACTTCAGATCGTTTCATTTGCTTGTTATCCATGAATCTCTCATCATCATACTGAGACTTGATTCCTTCTTTATCAGGTACAAGAGCTGGAGGAGGCAGTGAGCAATGACAATGCAGCTGCTGAACATCAGGAGACACCAGTTGACTCGTGGATGGTAGGTTCAGTTCACTGAATGGATCAATCCATTTCACATCAGATCTTTCAGATGGAAGTGAATGTGTTTTAGTCACAAAAGTGCCCTGAAGCTCAGTTTACACAAGCAGAGAAAACAAACAGAGTAAGTTATGATGATGCTGATGAAGGTCTCCTCATGTCTCATGTCTCTCACACAGATGCCAAACAACAGACAGAAGCGTGGCTTTAAGTGTAAGTTCTGCTGCGGCTGCTGCAGAGCTGGTGTCTGTGGACTGTGCTGCAAATTCTGAGGACCTGCCAGCA // NRC217 (SEQ ID NO: 151)AAGATGAAGACATCAGTGGTTGCAGTCACAGTGGCCGTCGTGCTCGTCTTTATTTGTATCCAGCAGAGCTCTGCCACCTTTCCTGAGGTAAGCACCTGACTTCAGATAGCTTCATTTGCTTGTTATCCATGAATCTCTCATCAACATACTGAGACTTTATTCCTTCTTTATCAGGTACAAGAGCTGGAGGAGGCAGTGAGCAATGACAATGCAGCTGCTGCGCATCAGGAGACATCAGTGGACTCGTGGATGGTAGGTTCAGTTCACTCAATGGATCAAACCAATTCACATCAGATCTTTCAGATGGAAGCGAATGTGTTTTAGTCAAAAAAGTGACCTGATGCTCAGTTTACACAAGCAGAGAAAACAAGCAGAGTAAGTTATGATGATGCTGATGAACGTGTCCTCATGTCTCATGTCTCTCACACAGATGCCATACAACAGACCGAAGCGTAGCTTTAAGTGTAAGTTCTGCTGCGGCTGCTGCAGAGCTGGTGTCTGTGGACTGTGCTGCAAATTCTGAGGATTCCTGCTCCAACAAC // NRC218 (SEQ ID NO: 152)AAGATGAAGACATTCAGTGTGGCAGTCACAGTGGCCGTCGTGCTCGTCTTTATTTGTATCCAGCAGAGCTCTGCCACCTTTCCTGAGGTAAGCACCTGACTTCAGATAGCTTCATTTGCTTGTTATCCATGAATCTCTCATCAACATACTGAGACTTGATTTCTTCTTTATCAGGTACAAGAGCTGGAGGAGGCAGTGAGCAATGACAATGCAGCCGCTGAACATCAGGAGACATCAGTGGACTCGTGGATGGTAGGTTCAGTTCACTCAATGGATCAAACCAATTCACATCAGATCTTTCAGATGGAAGTGAATGTGTTTTAGTCACAGAAGTGCCCTGATGCTCAGTTTACACAAGCAGAGAAAACAAGCAGAGTAAGTTATGATGATGCTGATGAACGTGTCCTCATGTCTCATGTCTCTCACACAGATGCCATACAACAGACCGAAGCGTAGCTTTAAGTGTAAGTTCTGCTGCGOCTGCTGTAGAGCTGGTGTCTGTGGACTGTGCTGCAAATTCTGAGGATTCCTGCTCCAACAAC // NRC219 (SEQ ID NO: 153)AAGATGAAGACATTCGTGGTTGCAGTCACAGTGGCCGTCGTGCTCGTCTTTATTTGTATCCAGCAGAGCTCTGCCACCTTTCCTGAGGTAAGCACCTGACTTCAGATAGCTTCATTTGCTTGTTATCCATGAATCTCTCATCAACATACTGAGACTTGATTCCTTCTTTATCAGGTACAAGAGCTGGAGGAGGCAGTGAGCAATGACAATGCAGCCGCTGAACATCAGGAGACATCAGTGGACTCGTGGATGGTAGGTTCAGTTCACTCAATGGATCAAACCAATTCACATCAGATCTTTCAGATGGAAGTGACTGTGTTTTAGTCACAAAAGTGCCCTGATGCTCAGTTTACACAAGCAGAGAAAACAAGCAGAGTAAGTTATGATGATGCTGATGAACGTCTCCTCATGTCTCATGTCTCTCACACAGATGCCATACAACAGACAGAAGCGTAGCTTTAAGTGTAAGTTCTGCTGCGGCTGCTGCAGAGCTGGTGTCTGTGGACTGTGCTGCAAATTCTGAGGATTCCTGCTCCAACAAC // NRC220 (SEQ ID NO: 154)AAGATGAAGACATCAGTGGTTGCAGTCACAGTGGCCGTCGTGCTCGTCTTTATTTGTATCCAGCAGAGCTCTGCCACCTTTCCTGAGGTAAGCACCTGACTTCAGATAGCTTCATTTGCTTGTTATCCATGAATCTCTCATCAACATACTGAGACTTTATTCCTTCTTTATCAGGTACAAGAGCTGGAGGAGGCAGTGAGCAATGACAATGCAGCTGCTGCACATCAGGAGACATCAGTGGACTCGTGGATGGTAGGTTCAGTTCACTCAATGGATCAAACCAATTCACATCAGATCTTTCAGATGAAGTGACTGTGTTTTAGTCACAAAAGTGCCCTGATGCTCAGTTTACACAAGCAGAGAAAACAAGCAGAGTAAGTTATGATGATGCTGATGAACGTGTCCTCATGTCTCATGTCTCTCACACAGATGCCATACAACAGACATAAGCGTAGCTTTAAGTGTAAGTTCTGCTGCGGCTGCTGCAGAGCTGGTGTCTGTGGACTGTGCTGCAAATTCTGAGGATTCCTGCT // NRC221 (SEQ ID NO: 155)AAGATAAGACATTCAGTGTTGCAGTCACAGTGGCCGTCGTGCTCGTCTTTATTTGTATCCAGCAGAGCTCTGCCACCTTTCCTGAGGTAAGCACCTGACTTCAGATCGTTTCATTTGCTTGTTAGCCTTGAATCTCTCATCAACATACTGAGACTTGATTTCTTCTTTATCAGGTACAAGAGCTGGAGGAGGCAGTGAGCAATGACAATGCAGCTGCTGAACATCAGGAGACATCAGTGGACTTGTGGATGGTAGGTTCAGTTCACTGAATGGATCAAACCAATTCACATCAGATCTTTCAGATGGAAGTGAATGTGTTTTAGTCACAAAAGTGCCCTGAAGCTCAGTTTACACGAGCAGAGAAAACCAACACAGTAAGTTATGATGATGCTGATGAACGTCTCCTCATGTCTCATGTCTCTCACACAGATGCCATACAACAGACAGAAGCGTGGCTTTAAGTGTAAGTTCTGCTGCGGCTGCTGCAGCCCTGGTGTCTGTGGACTTTGCTGCAGATTCTGAGGATTCCTGCTCCAACAAC // NRC222 (SEQ ID NO: 156)AAGATGAAGACATTCAGTGTTGCAGTCGCAGTGGCCGTCGTGCTCATCTTTATTTGTATCCAGCAGAGCTCTGCCACCTTTCCTGAGGTAAGCACCTGACTTCAGATAGTTTCATTTGCTTGTTATCCATGAATCTCTCATCAACATACTGAGACTTTATTCCTTCTTTATCAGGTACAAGAGCTGGAGGAGGCAGTGAGCAATGACAATGCAGCTGCTGAACATCAGGAGACATCATTGGACTCATGGATGGTAGGTTCAGTTCACTCAATGGATCAAACCAATTCACATCAGATCTTTCAGATGGAAGTGACTGTGTTTTAGTCACAAAAGTGCCCTGATGCTCAGTTTACACAAGCAGAGAAAACAAGCAGAGTAAGTTATGATGATGCTGATGAACGTGTCCTCATGTCTCATGTCTCTCACACAGATGCCATACAACAGACAGAAGCGTGGCTTTAAGTGTAAGTTCTGCTGCGGCTGCTGCAGAGCTGGTGTCTGTGGACTGTGCTGCAAATTCTGAGGACCTGCCAGCA // NRC223 (SEQ ID NO: 157)AAGATGAAGACATTCAGTGTTGCAGTCACAGTGGCCGTCGTGCTCGTCTTTATTTGTATCCAGCAGAGCTCTGCCACCTTTCCTGAGGTAAGCACCTGACTTCAGATAGTTTCATTTGCTTGTTATCCATGAATCTCTCATCAACATACTGAGACTTTATTCCTTCTTTATCAGGTACAAGAGCTGGAGGAGGCAGTGAGCAATGACAATGCAGCTGCTGAACATCAGGAGACATCATTGGACTCATGGATGGTAGGTTCAGTTCACTCAATGGATCAAACCAATTCACATCAGATCTTTCAGATGGAAGTGAATGTGTTTTAGTCACAAAAGTGCCCTGATGCTCAGTTTACACAAGCAGAGAAAACAAGCAGAGTAAGTTATGATGATGCTGATGAACGTGTCCTCATGTCTCATGTCTCTCACACAGATGCCATACAACAGACATAAGCGTAGCTTTAAGTGTAAGTTCTGCTGCGGCTGCTGCAGAGCTGGTGTCTGTGGACTGTGCTGCAAATTCTGAGGACCTGCCAGCA // NRC224 (SEQ ID NO: 158)AGATGAAGACATTCAGTGTTGCAGTCACAGTGGCCGTCGTGCTCGTCTTTATTTGTATCCAGCAGAGCTCTGCCACCTTTCCTGAGGTAAGCACCTGACTTCAGATAGTTTCATTTGCTTGTTATCCATGAATCTCTCATCAACATACTGAGACTTGATTTCTTCTTTATCAGGTACAAGAGCTGGGGGAGGCAGTGAGCAATGACAATGCAGCCGCTGAACATCAGGAGACATCAGTGGACTCGTGGATGGTAGGTTCAGTTCACTCAATGGATCAAACCAATTCACATCAGATCTTTCAGATGGAAGTGAATGTGTTTTAGTCACAAAAGTGCCCTGATGCTCAGTTTACACAAGCAGAGAAAACAAGCAGAGTAAGTTATGATGATGCTGATGAACGTGTCCTCATGTCTCATGTCTCTCACACAGATGCCATACAACAGACCGAAGCGTAGCTTTAAGTGTAAGTTCTGCTGCGGCTGCTGCAGAGCTGGTGTCTGTGGACTGTGCTGCAAATTCTGAGGACCTGCCAGCA // NRC225 (SEQ ID NO: 159)AAGATGAAGACATTCAGTGTTGCAGTCACAGTGGCCGTCGTGCTCATCTTTATTTGTATCCAGCAGAGCTCTGCCACCTCTCCTGAGGTACAAGGGCTGGAGGAGGCAGTGAGCAATGACAATGCAGCTGCTGAACATCAGGAGACATCAGTGGACTCGTGGATGATGCCATACAACAGACAGAAGCGTGGCTTTAAGTGTAAGTTCTGCTGCGGCTGCTGCAGGCCTGGTGTCTGTGGACTTTGCTGCAGATCCTGAGGATTCCTGCTCCAACAAC //NRC226 (SEQ ID NO: 160)AAGATGAAGACATTCAGTGTTGCAGTCACAGTGGCCGTCGTGCTCGTCTTTATTTGTATCCAGCAGAGCTCTGCCACCTTTCCTGAGGTAAGCACCTGACTTCAGATCGTTTCATTTGCTTGTTAGCCTTGAATCTCTCATCAACATACTGAGACTTGATTTCTTCTTTATCAGGTACAAGAGCTGGAGGAGGCAGTGAGCAATGACAATGCAGCTGCTGAACATCAGGAGACATCAGTGGACTTGTGGATGGTAGGTTCAGTTCACTGAATGGATCAAACCAATTCACATCAGATCTTTCAGATGGAAGTGAATGTGTTTTAGTCACAAAAGTGCCCTGAAGCTCAGTTTACACGAGCAGAGAAAACCAACACAGTAAGTTATGATGATGCTGATGAACGTCTCCTCATGTCTCATGTCTCTCACACAGATGCCATACAACAGACAGAAGCGTGGCTTTAAGTGTAAGTTCTGCTGCGGCTGCTGCAGACCTGGTGTCTGTGGACTTTGCTGCAGATTCTGAGGATTCCTGCTCCAACAAC // NRC227 (SEQ ID NO: 161)AAGATGAAGACATTCAGTGTTGCAGTCACAGTGGCCGTCGTGCTCGTCTTTATTTGTATCCAGCAGAGCTCTGCCACCTTTCCTGAGGTAAGCACCTGACTTCAGATCGTTTCATTTGCTTGTTAGCCTTGAATCTCTCATCAACATACTGAGACTTGATTTCTTCTTTATCAGGTACAAGAGCTGGAGGAGGCAGTGAGCAATGACAATGCAGCTGCTGAACATCAGGAGACATCAGTGGACTTGTGGATGGTAGGTTCAGTTCACTGAATGGATCAAACCAATTCACATCAGATCTTTCAGATGGAAGTGAATGTGTTTTAGTCACAAAAGTGCCCTGAAGCTCAGTTTACACGAGCAGAGAAAACCAACACAGTAAGTTATGATGATGCTGATGAACGTCTCCTCATGTCTCATGTCTCTCACACAGATGCCATACAACAGACAGAAGCGTGGCTTTAAGTGTAAGTTCTGCTGCGGCTGCTGCAGTCCTGGTGTCTGTGGAGTTTGCTGCAGATTCTGAGGATTCCTGCTCCAAC // NRC228 (SEQ ID NO: 162)AAGATGAAGACATTCAGTGTTGCAGTCACAGTGGCCGTCGTGCTCGTCTTTATTTGTATCCAGCAGAGCTCTGCCACCTTTCCTGAGGTAAGCACCTGACTTCAGATCGTTTCATTTGCTTGTTAGCCTTGAATCTCTCATCAACATACTGAGACTTGATTTCTTCTTTATCAGGTACAAGAGCTGGAGGAGGCAGTGAGCAATGACAATGCAGCTGCTGAACATCAGGAGACATCAGTGGACTCGTGGATGGTAGGTTCAGTTCACTGAATGGATCAAACCAATTCACATCAGATCCTTCAGATGGAAGTGAATGTGTTTTAGTCACAAAAGTGCCCTGAAGCTCAGTTTACACGAGCAGAGAAAACAAACACAGTAAGTTATGATGATGCTGATGAACGTCTCCTCATGTCTCATGTCTCTCACACAGATGCCATACAACAGACAGAAGCGTGGCTTTAAGTGTAAGTTCTGCTGCGGCTGCTGCAGTCCTGGTGTCTGTGGACTTTGCTGCAAATTCTGAGGACCTGCCAGCA // NRC229 (SEQ ID NO: 163)AAGATGAAGACATTCAGTGTTGCAGTCACAGTGCCCGTCGTGCTCGTCTTTATTTGTATCCAGCAGAGCTCTGCCACCTTTCCTGAGGTAAGCACCTGACTTCAGATCGTTTCATTTGCTTGTTAGCCTTGAATCTCTCATCAACATACTGAGACTTGATTTCTTCTTTATCAGGTACAAGAGCTGGAGGAGGCAGTGAGCAATGACAATGCAGCTGCTGAACATCAGGAGACATCAGTGGACTCGTGGATGGTAGGTTCAGTTCACTGAATGGATCAAACCAATTCACATCAGATCTTTCAGATGGAAGTGAATGTGTTTTAGTCACAAAAGTGCCCTGAAGCTCAGTTTACACGAGCAGAGAAAACAAACACAGTAAGTTATGATGATGCTGATGAACGTCTCCTCATGTCTCATGTCTCTCACACAGATGCCATACAACAGACAGAAGCGTGGCTTTAAGTGTAAGTTCTGCTGCGGCTGCTGCAGACCTGGTGTCTGTGGACTTTGCTGCAAATTCTGAGGACCTGCCAGCA // NRC230 (SEQ ID NO: 164)AAGATGAAGACATTCAGTGTTGCAGTCACAGTGGCCGTCGTGCTCGTCTTTATTTGTATCCAGCAGAGCTCTGCCACCTTTCCTGAGGTACAAGAGCTGGAGGAGGCAGTGAGCAATGACAATGCAGCTGCTGAACATCACGAGACATCAGTGGACTCGTGGATGATGCCATACAACAGACAGAAGCGTGGCTTTAAGTGTAAGTTCTGCTGCGGCTGCTGCAGACCTGGTGTCTGTGGACTTTGCTGCAAATTCTGAGGACCTGCCAGCA // NRC231(SEQ ID NO: 165)AAGATGAAGACATTCAGTGTTGCAGTCACAGTGGCCGTCGTGCTCGTCTTTATTTGTATCCAGCAGAGCTCTGCCACCTTTCCTGAGGTACAAGAGCTGGAGGAGGCAGTGAGCAATGACAATGCAGCTGCTGAACATCAGGAGACATCAGTGGACTCGTGGATGATGCCATACAACAGACAGAAGCGTGGCTTTAAGTGTAAGTTCTGCTGCGGCTGCTGCAGGCCTGGTGTCTGTGGACTTTGCTGCAGATTCTGAGGATTCCTGCTCCAACAAC //NRC232 (SEQ ID NO: 166)AAGATGAAGACATTCAGTGTTGCAGTCACAGTGGCCGTCGTGCTCGTCTTCATTTGTATCCAGCAGAGCTCTGCCACCTTTCCTGAGGTAAGCACCTGACTTCAGATCGTTTCATTTGCTTGTTAGCCTTGAATCTCTCATCAACATACTGAGACTTGATTTCTTCTTTATCAGGTACAAGAGCTGGAGGAGGCAGTGAGCAGTGACAATGCAGCTGCTGAACATCAGGAGACATCAGTGGACTCGTGGATGGTAGGTTCAGTTCACTGAATGTGTTTTAGTCACAAAAGTGCCCTGAAGCTCAGTTTACACAAGCAGAGAAAACAAACAGAGTAAGTTATGATGATGCTGATGAACGTCTCCTCATGTCTCATGTCTCTCACACAGATGCCATACAACAGACAGAAGCGTAGCTTTAAGTGCAAGTTCTGCTGCGGCTGCTGCAGACGTGGTGTCTGTGGACTGTGCTGCAAATTCTGAGGATTCCTGCTCCAACAAC // NRC233 (SEQ ID NO: 167)AAGATGAAGACTATCAGTGTTGCAGTCACAGTGGCCGTCGTGCTCCTCTTCATTTGTACCCAGCAGAGCTCTGCCACCTTTCCTGAGGTAAGCACCTGACTTCAGATCGTTTCATTTGCTTGTTAGCCTTGAATCTCTCATCAACATACTGAGACTTGATTTCTTCTTTATCAGGTACAAGAGCTGGAGGAGGCAGTGAGCAGTGACAATGCGGCTGCTGAACATCAGGAGACATCAGTGGACTCGTGGATGGTAGGTTCAGTTCACTGAATGGATCAAACCAATTCACATCAGATCTTTCAGATGOAAGTGAATGTGTTTTAGTCACAAAAGTGCCCTGAAGCTCAGTTTACACAAGCAGAGAAAACAAACACAGTAAGTTATGATGATGCTGATGAACGTCTCCTCATGTCTCATGTCTCATGTCTCTCACACAGATGCCATACAACAGACAGAAGCGTGGCTTTAAGTGCAAGTTCTGCTGCGGCTGCCGCTGTGGTGCTCTCTGTGGACTGTGCTGCAAATTCTGAGGATTCCTGCTCCAACAAC // NRC234 (SEQ IDNO: 168)AAGATGAAGACATTCAGTGTTGCAGTCACAGTGGCCGTCGTGCTCGTCTTCATTTGTATCCAGCAGAGCTCTGCCACCTTTCCTGAGGTAAGCACCTGACTTCAGATCGTTTCATTTGCTTGTTAGCCTTGAATCTCTCATCAACGTACTGAGACTTGATTTCTTCTTTATCAGGTACAAGAGCTGGAGGAGCCAGTGAGCAGTGACAATGCAGCTGCTGAACATCAGGAGACATCGGTGGACTCGTGGATGGTAGGTTCAGTTCACTGAATGGATCAAACCAATTCACATCAGATCTTTCAGATGGAAGTGAATGTGTTTTAGTCACAAAAGTGCCCTGAAGCTCAGTTTACACAAGCAGAGAAAACAAACACAGTAAGTTATGATGATGCTGATGAACGTCTCCTCATGTCTCATGTCTCATGTCTCTCACACAGATGCCATACAACAGACAGAAGCGTGGCTTTAAGTGCAAGTTCTGCTGCGGCTGCCGCTGTGOTGCTCTCTGTGGACTGTGCTGCAAATTCTGAGGACCTGCCAGCA // NRC235 (SEQ ID NO:169)AAGATGAAGACATTCAGTGTTGCAGTCACAGTGGCCGTCGTGCTCGTCTTTATTTGTTCCAGCAGAGCTCTGCCACCTTTCCTGAGGTAAGCACCTGACTTCAGATCGTTTCATTTGCTTGTTAGCCTTGAATCTCTCATCAACATACTGAGACTTGATTTCTTCTTTATCAGGTACAAGAGCTGGAGGAGGCAGTGAGCAGTGACAATGCAGCTGCTGAACATCAGGAGACATCAGTGGACTCGTGGATGGTAGGTTCAGTTCCCTGAATGGATCAAACCAATTCACATCAGATCTTTCAGATGGAAGTGAATGTGTTTTAGTCACAAAAGTGCCCTGAAGCTCAGTTTACACAAGCAGAGAAAACAAACACAGTAAGTTATGATGATGCTGATGAACATCTCCTCATGTCTCATGTCTCATGTCTCTCACACAGATGCCATACAACAGACAGAAGCGTGGCTTTAAGTGCAAGTTCTGCTGCGGCTGCCGCTGTGGTGCTCTCTGTGGACTGTGCTGCAAATTCTGAGGACCTGCCAGCA // NRC236 (SEQ ID NO: 170)ACGAGCTGACAGGAGCTGACAGGAGTCACCAGCAGAGTCAAAGAACTAAACAACTTAACTCAGTCAAACTCTCAAAGATGAAGACATTCAGTGTTGCAGTCACAGTGGCCGTCGTGCTCGTCTTTATTTGTATCCAGCAGAGCTCTGCCACCTTTCCTGAGGTACAAGAGCTGGAGGAGGCAGTGAGCAATGACAATGCAGCTGCTGAGCATCAGOAGACACCAGTGGACTCAGGGATGATGCCAAACAACAGACAGAAGCGCAGCGCCGATTGTTGGCCATGTTGCAATCAAAATGGCTGTGGAACTTGCTGCAAGGTCTAAACAGACTCTTGGGCAGATCAATCCAGGTTCGTCTTTCGTTGTCTCTCCGTGGAGTCGAACCAGAGACCTTCTCAGCCCATAGTCCAAGTTTCTGCCACTAGACCACCGCCTCTCCCTCATCAAATACTCAATGTTTTTCATTTTGTCTTAAAGTTCATTGAACTATAAACATATTTCTGGTAGAGCATGTGATAGTTTAATGGTGTTACTCATTGGTTCATGGTATAGTCAGATGTTCAGAGATGTGATTATATCATCCACATATTTTCTCTGTTAAGGTGTACTGTCAATAAATGTCAATGCTCCTTTGAAAAAAAAAAAAAAAAAAAAAC // NRC237 (SEQ ID NO: 171)CGTGCTCGTCTTTATTTGTATCCAGCAGAGCTCTGCCACCTTTCCTGAGGTGAGCTCCTGACTTCAGATCGTTTCATTTAGCTTGTTATCCATGAATCTCTCATCAACATACTGAGACTTGAATCCTTCTTTATCAGGTACAGGAGCTGGAGGAGGCAGTGAGCAATGACAATGCAGCTGCTGAACATCAGGAGACATCAGTGGACTCATGGATGGTATGTTCAGTTCACTGAATGGATCAAACCAATTCACATCAGATCTTTCAGATGGAAGTGAATTTGTTTTAGTCCCAAAAGTGCCCTGAAGCTCAGTTTACACAAGCAGAGAAAAACAAAACACAGTAAGTTATGATGATGCTGATGAACGTCTCCTCATGTCTCATGTCTCTCACACAGATGCCATACAACAGACAGAAGCGCAGCGCCGAGTGTAGCTTCTGCTGCAATGAATCTGGCTGTGGAATTTGCTGCAAATTCTGAGGATTCCTGCTCCAACAACAAGGGCGAATTC // NRC238 (SEQ ID NO: 172)AAGATGAAGACATTCAGTGTTGCAGTCACAGTGGCCGTCGTGCTCGTCTTTATTTGTATCCAGCAGAGCTCTGCCACCTTTCCTGAGGTGAGCTCCTGACTTCAGATCGTTTCATTTAGCTTGTTATCCATGAATCTCTCATCAACATACTGAGACTTGAATCCTTCTTTATCAGGTACAGGAGCTGGAGGAGGCAGTGAGCAATGACAATGCAGCTGCTGAACATCAGGAGACATCAGTGGACTCATGGATGGTATGTTCAGTTCACTGAATGGATCAAACCAATTCACATCAGATCTTTCAGATGGAAGTGAATTTGTTTTAGTCCCAAAAGTGCCCTGAAGCTCAGTTTACACAAGCAGAGAAAAACAAAACACAGTAAGTTATGATGATGCTGATGAACGTCTCCTCATGTCTCATGTCTCTCACACAGATGCCATACAACAGACAGAAGCGCAGCGCCGAGTGTAGCTTCTGCTGCAATGAATCTGGCTGTGGAATTTGCTGCAAATTCTGAGGACCTGCCAGCA // NRC239 (SEQ ID NO: 173)GTGGAGGAGCCAGTGAGCAGTGAGAATGGAGCAAATGAACACACATAAGATCTTTCGGATGGAAGTGTATGTGTTTTAGTCACATGAGTGGCTCGAAGCTCAGTACACACGAGCAGAGAGAACGAACACAGTGTGTTTTATTCTGCTTGTGTAAACTGAGCTTCAGTTTACACAAGCAGAGAAAACAAACACAGTAAGTTATGATGATGCTGATGAACGTCTCCTCATGTCTCATATCTCTCACACAGATGCCAAACAACAGACAGAAGCGTGGCTCTAATTGCAAACCATGCTGCAATCATAATGGCTGTGGAACGTGCTGCGAAGTCTGAGGATTCCTGCTCCACA //

1-18. (canceled) 19-42. (canceled)
 43. A method of screening a testnucleic acid sequence to identify a candidate nucleic acid sequenceencoding an antimicrobial peptide, said method comprising: (a)identifying an initial peptide of interest; (b) identifying a DNAsequence from a first fish species containing a nucleotide sequenceencoding the initial peptide; (c) identifying within the DNA sequence aflanking nucleotide sequence on each side of the peptide-encodingsequence; (d) obtaining a primer oligonucleotide sequence complementaryto each flanking sequence; and (e) screening a test nucleic acidsequence from a fish species other than the first fish species todetermine whether it is capable of being amplified by PCR using theprimers from step (d); amplification indicating that the test nucleicacid sequence is a candidate nucleic acid sequence encoding anantimicrobial peptide.
 44. The method of claim 43 wherein the initialpeptide has a net positive charge of at least 2 and has an amphipathicstructure.
 45. The method of claim 43 wherein the initial peptide isselected from the group consisting of a hepcidin, a pleurocidin, apardaxin, a misgurin, HFA-1, a piscidin, a moronecidin, and a cleavageproduct of histone 2A from catfish.
 46. The method of claim 45 whereinthe cleavage product of histone 2A is a parasin.
 47. The method of claim43 comprising a further step (f) of predicting the amino acid sequenceencoded by the candidate sequence and selecting nucleic acid sequenceswhich are predicted to encode peptides having an amphipathic structureand a net charge.
 48. The method of claim 47 comprising a furtheradditional step of obtaining a peptide corresponding to the candidatenucleic acid sequence and assaying the peptide sequence forantimicrobial activity.
 49. The method of claim 43 comprising a furtherstep (a′) of confirming that the initial peptide has antimicrobialactivity.
 50. The method of claim 43 wherein the initial peptide is apleurocidin.
 51. The method of claim 50 wherein at least one of theflanking sequences is selected from the group consisting of a nucleotidesequence encoding signal sequence I (SEQ ID NO: 305), a nucleotidesequence encoding Acidic Sequence I (SEQ ID NO: 306),GCCCACTTTGTATTCGCAAG (SEQ ID NO: 5) and CTGAAGGCTCCTTCAAGGCG. (SEQ IDNO: 6)


52. The method of claim 43 wherein the initial peptide is a hepcidin.53. The method of claim 52 wherein at least one flanking sequence isselected from the group consisting of a nucleotide sequence encodingsignal peptide II (SEQ ID NO: 307), a nucleotide sequence encodingsignal peptide III (SEQ ID NO: 308), a nucleotide sequence encodingsignal peptide IV (SEQ ID NO: 309), a nucleotide sequence encodingsignal peptide V (SEQ ID NO: 310), a nucleotide sequence encodingprosequence I (SEQ ID NO: 311), a nucleotide sequence encodingprosequence II (SEQ ID NO: 312), ACAACCTCGTCCTTAGG (SEQ D NO: 313) andACGCCCGTCCAGGAAT. (SEQ ID NO: 314)


54. An isolated nucleic acid sequence identifiable using the method ofclaim
 43. 55. The nucleic acid sequence of claim 54 wherein the sequenceis selected from the group consisting of SEQ ID NOS: 82-124, 129-173 and327.
 56. An isolated polypeptide capable of being encoded by the nucleicacid sequence of claim
 54. 57. A kit comprising: a. a first nucleic acidsequence at least 95% identical to a first flanking sequence, located ator near a 5′ end of a target sequence encoding an antimicrobial peptide;b. a second nucleic acid sequence at least 95% identical to a secondflanking sequence located at or near a 3′ end of a target sequenceencoding an antimicrobial peptide; and c. instructions for carrying outthe method of claim
 43. 58. The method of claim 43, wherein at least onesequence selected from the group consisting of signal sequence I, acidicsequence I, signal peptide II, signal peptide III, signal peptide IV,signal peptide V, prosequence I, prosequence II, nucleic acid sequencesencoding them, and nucleic acid sequences substantially complementary tosuch encoding nucleic acids, is identified.
 59. An isolatedantimicrobial peptide at least 80% homologous to one of peptide a, b, cor d: Peptide a GW(G/K)XXFXK Peptide b GXXXXXXXHXGXXIH Peptide cFKCKFCCGCCXXGVCGXCC Peptide d CXXCCNCC(K/H)XKGCGFCCKF Peptide eFKCKFCCGCRCGXXCGLCCKF Peptide f XXXCXXCCNXXGCGXCCKX


60. The antimicrobial peptide of claim 59 which is at least 90%homologous to one of peptide a, b, c or d.
 61. The antimicrobial peptideof claim 59 which is one of peptide a, b, c or d.
 62. A method ofscreening a test nucleic acid sequence to identify a candidate nucleicacid sequence encoding an antimicrobial peptide, said peptidecomprising: a) identifying a nucleic acid sequence encoding an initialpeptide of interest; (b) identifying a DNA sequence from a first fishspecies containing a nucleotide sequence encoding the initial peptide;(c) identifying within the DNA sequence a flanking nucleotide sequenceon each side of the peptide-encoding sequence; (d) obtaining a primeroligonucleotide sequence complementary to each flanking sequence; and(e) screening a test nucleic acid sequence from a fish species otherthat the first fish species to determine whether it is capable of beingamplified by PCR using the primers from step (d); amplificationindicating that the test nucleic acid sequence is a candidate nucleicacid sequence encoding an antimicrobial peptide.
 63. An isolatedantimicrobial peptide selected from the group consisting of: (a)WLRRIGKGVKIIGGAALDHL; (b) GRRKRKWLRRIGKGVKIIGGAALDHL; (c)RWGKWFKKATHVGKHVGKAALTAYL; (d) RSTEDIIKSISGGGFLNAMNA; (e)FFRLLFHGVHHGGGYLNAA; (f) FFRLLFHGVHHVGKIKPRA; (g)GWKSVFRKAKKVGKTVGGLALDHYL; (h) GWKKWFNRAKKVGKTVGGLAVDHYL; (i)GWRTLLKKAEVKTVGKLALKHYL; (j) AGWGSIFKHIFKAGKFIHGAIQAHND; (k)GFWGKLFKLGLHGIGLLHLHL; (l) GWKKWLRKGAKHLGQAAIK; (m)GWKKWLRKGAKHLGQAAIIKGLAS; (n) GWKKWFTKGERLSQRHFA; (o)FLGLLFHGVHHVGKWIHGLIHGHH; (p) GFLGILFHGVHHGRKKALHMNSERRS; (q)FLGFLFHGIHHGIRAIHLIHG; (r) FFGALIKGAIHGGKLLHKLIKKKHEHHGYGKHWG; (s)FLGFLFHGIRHGIKAIHGMIHG; (t) GKGRWLERIGKAGGIIIGGALDHLG; (u)GLGNWMGPHISGEKKALHMNSERRS; (v) GLGNWIVRPIGGEKKALQMNSERRS; (w)LFGKFLKKVVHAGTSIGETALHVAAEHHGLHAHHG; (x) GLGNWMGPHISGRKKALHMNSERRS; (y)FLGLLFHGVHHVGKLIHGLIHG; (z) ARWGTFFKIFKAGRFIHGAIQAHNDG; (aa)AWIPALNRIYHGALLRINRQMVYYRRHWHG; (ab) AWMPALNRIYHGALLRINRQMVYYRRHWHG;(ac) GWKKWFTKGAKHLGQAAINGLAS; (ad) GWKKWLRKGAKHLGQAALKGLAS; (ae)FGDFYMKPGRKISHGYIRSPYG; (af) GYWRFRNHRGERLSQRHFA; (ag)FGMLFHRVHHAGRLIHRFIKRHG; (ah) IFGLIATAVHNAGRLIHRLLGFHHGPPGFWHG; (ai)IFGLIATAVHNVGRLVHGLLGFHHGPPGFWHG; (aj) IFGLIATAVHNVGRLVHGLLGFHHGPPRFWHG;(ak) FFGMRFHGVHHAGGGFLNAQGLLPSLLLNPGYRG; (al) FFGALLKGAQALHGIIHNARHG;(am) GWKDWFRKAKKVGKTVGGLALNHYLG; (an) GIRKWFKKAAHVGKEVGKVALNACL; (ao)GLKLKWFKKAVHVGKKVGKVALNAYLG; (ap) GWRKWIKKATHVGKHIGKAALDAYIG; (aq)GCKKWFKKAAHVGKNVGKVALNAYLG; (ar) GIRKWFKKAAHVGKKVGKVALNAYLG; (as)WLERKWFKKATHVGKHVGKAALDAYLG; (at) FFGLLFHGIHHAGKLIHGLIHHG; (au)LGNWMGPHISGRKKALQMNSERRS; (av) FLGLLFHGVHHVGNLIHGLIHHG; (aw)GIRKWFKKAAHVGKKVGKVALNAYLG;

(ax) a C-terminally amidated or otherwise C-terminally or N-terminallymodified peptide of (a) to (z) or (aa) to (aw); (ay) a C-terminallyamidated peptide of (a) to (z) or (aa) to (aw) where modificationreplaces C-terminal G; and (az) a peptide of (a) to (z) or (aa) to (aw)comprising at least one conservative amino acid substitution or deletionof an amino acid residue thereof.
 64. An isolated nucleotide sequenceencoding a peptide of claim
 63. 65. An isolated antimicrobial peptideselected from the group consisting of: (a)MKTFSVAVAVVVVLACMFILESTAVPFSEVRTEEVESIDSPVGE-HQQPGGTSMNLPMHFRFKRQSHLSLCRWCCNCCHNKGCGFCCKF; (b)MKTFSVAVAVVVVLACMFILESTAVPFSEVRTEEVESIDSPVGE-HQQPGGTSMNLPMHFRFKRQSHLSLCRWCCNCCHNKGCGFCCKF; (c)MKAFSVAVVLVIACMFILESTAVPFSEVRTEEVGSFDSPVGEHQ-QPGGESMHLPEPFRFKRQIHLSLCGLCCNCCHNIGCGFCCKF; (d)RTEEVESIDSPVGEHQQPGGTSMNLPMHFRFKRQSHLSLCRWCC- NCCHNKGCGFCCKF; (e)MKTFSVAVVPVIACMFILESTAVPFSEVRTEEVGSFDSPVGEHQ-QPGGTSMNLPMHFRFKRQSHLSLCRWCFNCCHNKGCGFCCKF; (f)MKQFSVAVVLVMACMFIVESTAVPFSEVRTEEVGSLDSPVGEHQ-QPGGESMHLPEPFRFKRQIHLSLCGLCCNCCHNIGCGFCCKF; (g)MKAFSIAVAVTLVLAFVCIQCSSAVPFQGVQELEEAGGNDTPVA-EHQVMSMESWMENPTRQKRHISHISLCRWCCNCCKANKGCGFCC- KF; (h)MKTFSVAVAVTLVLAFVCIQDSSAVPFQGVQELEEAGGNDTPVA-AHQMMSMESWMESPVRQKRHISHISMCRWCCNCCKAKGCGPCCK- F; (i)MKTFSVAVTVAVVLVFICIQQSSGTFPEVQELEEAVSNDNAAAE-HQETSVDSWMMPYNRQKRAFKCKCFCCGCCRAGVCGLCCKF; (j)MKTFSVAVTVAVVLVFICIQQSSASFPEAQELEEAVSNDNAAAE-HQETPVDSWMMPYNRQKRSFKCKFCCGCCRAGVCGLCCKF; (k)MKTFSVAVTVAVVLVFICIQQSSASFPEAQELEEAVSNDNAAAE-HQETPVDSWMMPNNRQKRGFKCKFCCGCCRAGVCGLCCKF; (l)MKTFSVAVTVAVVLVFICIQQSSATFPEMPYNRQKRGFKCKFCC- GCCGAGVCGMCCKF; (m)MKTFSVAVTVAVVLVFICIQQSSASFPEAQELEEAVSNDNAAAE-HQETPVDSRIPYNRQKRSFKCKFCCGCCRAGVCGLCCKF; (n)MKTCSVAVTVAVVLVFICIQQSSASFPEVQELEEAVSNDNAAAE-HQETPVDSWMMPNNRQKRGFKCKFCCGCCRAGVCGLCCKF; (o)MKTISVAVTVAVVLVFICIQQSSASFPEAQELEEAVSNDNAAAE-HQETPVDSGMIPYNRQKRSFKCKFCCGCCRAGVCGLCCKF; (p)MKTFSGAVTVAVVLVFICIQQSSASFPEVQELEEAVSNDNAAAE-HQETPVDSWMMPNNRQKRGFKCKFCCGCCRAGVCGLCCKF; (q)MKTSVVAVTVAVVLVFICIQQSSATFPEVQELEEAVSNDNAAAA-HQETSVDSWMMPYNRPKRSFKCKFCCGCCRA-GVCGLCCKF; (r)MKTFSVAVTVAVVLVFICIQQSSATFPEVQELEEAVSNDNAAAE-HQETSVDSWMMPYNRPKRSFKCKFCCGCCRAGVCGLCCKF; (s)MKTFVVAVTVAVVLVFICIQQSSATFPEVQELEEAVSNDNAAAE-HQETSVDSWMMPYNRQKRSFKCKFCCGCCRAGVCGLCCKF; (t)MKTSVVAVTVAVVLVFICIQQSSATFPEVQELEEAVSNDNAAAA-HQETSVDSWMMPYNRQKRSFKCKFCCGCCRAGVCGLCCKF; (u)MKTFSVAVTVAVVLVFICIQQSSATFPEVQELEEAVSNDNAAAE-HQETSVDLWMMPYNRQKRGFKCKFCCGCCSPGVCGLCCRF; (v)MKTFSVAVAVAVVLIFICIQQSSATFPEVQELEEAVSNDNAAAE-HQETSLDSWMMPYNRQKRGFKCKFCCGCCRAGVCGLCCKF; (w)MKTFSVAVTVAVVLVFICIQQSSATFPEVQELEEAVSNDNAAAE-HQETSLDSWMMPYNRHKRSFKCKFCCGCCRAGVCGLCCKLF; (x)MKTFSVAVTVAVVLVFICIQQSSATFPEVQELGEAVSNDNAAAE-HQETSVDSWMMPYNRPKRSFKCKFCCGCCRAGVCGLCCKF; (y)MKTFSVAVTVAVVLIFICIQQSSATSPEVQGLEEAVSNDNAAAE-HQETSVDSWMMPYNRQKRGFKCKFCCGCCRPGVCGLCCRS; (z)MKTFSVAVTVAVVLVFICIQQSSATFPEVQELEEAVSNDNAAAE-HQETSVDLWMMPYNRQKRGFKCKFCCGCCRPGVCGLCCRF; (aa)MKTFSVAVTVAVVLVFICIQQSSATFPEVQELEEAVSNDNAAAE-HQETSVDL-WMMPYNRQKRGFKCKFCCGCCSPGVCGLCCRF; (ab)KTFSVAVTVAVVLVFICIQQSSATFPEVQELEEAVSNDNAAAEH-QETSVDS-WMMPYNRQKRGFKCKFCCGCCSPGVCGLCCKF; (ac)MKTFSVAVTVAVVLVFICIQQSSATFPEVQELEEAVSNDNAAAE-HQETSVDS-WMMPYNRQKRGFKCKFCCGCCRPGVCGLCCKF; (ad)MKTFSVAVTVAVVLVFICIQQSSATFPEVQELEEAVSNDNAAAE-HQETSVDSWMMPYNRQKRGFKCKFCCGCCRPGVCGLCCKF; (ae)MKTFSVAVTVAVVLVFICIQQSSATFPEVQELEEAVSNDNAAAE-HQETSVDSWMMPYNRQKRGFKCKFCCGCCRPGVCGLCCRF; (af)MKTFSVAVTVAVVLVFICIQQSSATFPEVQELEEAVSSDNAAAE-HQETSVDSWMMPYNRQKRSFKCKFCCGCCRRGVCGLCCKF; (ag)MKTISVAVTVAVVLLFICTQQSSATFPEVQELEEAVSSDNAAAE-HQETSVDSWMMPYNRQKRGFKCKFCCGCRCGALCGLCCKF; (ah)MKTFSVAVTVAVVLVFICIQQSSATFPEVQELEEPVSSDNAAAE-HQETSVDSWMMPYNRQKRGFKCKFCCGCRCGALCGLCCKF; (ai)MKTFSVAVTVAVVLVFICIQQSSATFPEVQELEEAVSSDNAAAE-HQETSVDSWMMPYNRQKRGFKCKFCCGCRCGALCGLCCKF; (aj)MKTFSVAVTVAVVLVFICIQQSSATFPEVQELEEAVSNDNAAAE-HQETPVDSGMMPNNRQKRSADCWPCCNQNGCGTCCKV; (ak)MKTFSVAVTVAVVLVFICIQQSSATFPEVQELEEAVSNDNAAAE-HQETSVDSWMMPYNRQKRSAECSFCCNESGCGICCKF; (al)MKTFSVAVTVAVVLVFICIQQSSATFPEVQELEEAVSNDNAAAE-HQETSVDSWMMPYNRQKRSAECSFCCNESGCGICCKF; (am) MPNNRQKRGSNCKPCCNRNGCGTCCEV;(an) a C-terminally amidated peptide (a) to (z) or (aa) to (am); and(ao) a peptide of (a) to (z) or (aa) to (am) com- prising at least oneconservative amino acid substitution of an amino acid residue thereof.


66. An isolated nucleotide sequence encoding a peptide of claim 65.